Compositions and methods of modulating 15-pgdh activity

ABSTRACT

Compounds and methods of modulating 15-PGDH activity, modulating tissue prostaglandin levels, treating disease, diseases disorders, or conditions in which it is desired to modulate 15-PGDH activity and/or prostaglandin levels include 15-PGDH inhibitors and 15-PGDH activators described herein.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/943,932, filed Oct. 12, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/421,867, filed May 24, 2019 (now abandoned),which is a continuation of U.S. patent application Ser. No. 15/359,330,filed Nov. 22, 2016, (now U.S. Pat. No. 10,301,320 of May 28, 2019),which is a continuation of U.S. patent application Ser. No. 14/395,021,filed Oct. 16, 2014 (now U.S. Pat. No. 9,790,233 of Oct. 17, 2017),which is a U.S. national stage application under 35 U.S.C. § 371 ofInternational Application No. PCT/US2013/036790, filed Apr. 16, 2013,which in turn claims priority from U.S. Provisional Application No.61/624,670, filed Apr. 16, 2012, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.CA127306, CA095471, and CA150964 awarded by The National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

15-hydroyxy-prostaglandin dehydrogenase (15-PGDH) represents the keyenzyme in the inactivation of a number of active prostaglandins,leukotrienes and hydroxyeicosatetraenoic acids (HETEs) (e.g., bycatalyzing oxidation of PGE₂ to 15-keto-prostaglandin E2, 15k-PGE). Thehuman enzyme is encoded by the HPGD gene and consists of a homodimerwith subunits of a size of 29 kDa. The enzyme belongs to theevolutionarily conserved superfamily of short-chaindehydrogenase/reductase enzymes (SDRs), and according to the recentlyapproved nomenclature for human enzymes, it is named SDR36C1. Thus far,two forms of 15-PGDH have been identified, NAD+-dependent type I 15-PGDHand the type II NADP-dependent 15-PGDH, also known as carbonyl reductase1 (CBR1, SDR21C1). However, the preference of CBR1 for NADP and the highKm values of CBR1 for most prostaglandin suggest that the majority ofthe in vivo activity can be attributed to type I 15-PGDH.

Recent studies suggest that inhibitors of 15-PGDH and activators of15-PGDH could be therapeutically valuable. It has been shown that thereis an increase in the incidence of colon tumors in 15-PGDH knockoutmouse models. A more recent study implicates increased 15-PGDHexpression in the protection of thrombin-mediated cell death. It is wellknown that 15-PGDH is responsible for the inactivation of prostaglandinE2 (PGE₂), which is a downstream product of COX-2 metabolism. PGE₂ hasbeen found to be neurotoxic both in vitro and in vivo; thus, COX-2specific inhibitors, which decrease PGE₂ release, exhibitneuroprotective effects. PGE₂ has also been shown to be beneficial in avariety of biological processes, such as hair density, dermal woundhealing, and bone formation.

SUMMARY

Embodiments described herein relate to compounds and methods ofmodulating 15-PGDH activities, modulating tissue prostaglandin levels,and/or treating diseases, disorders, or conditions in which it isdesired to modulate 15-PGDH activity and/or prostaglandin levels.

In some embodiments, a 15-PGDH inhibitor can be administered to a tissueof a subject at an amount effective to increase prostaglandin levels inthe tissue. The 15-PGDH inhibitor can include formula (I):

-   -   wherein n is 0-2;    -   R₁ is a C₁₋₈ alkyl, which is linear, branched, or cyclic and        which is unsubstituted or substituted (e.g., R₁ can be a C₂₋₆        alkyl, C₂₋₄ alkyl, or C₄ alkyl, which is linear, branched, or        cyclic and which is unsubstituted or substituted);    -   R₂ and R₃ are the same or different and are each selected from        the group consisting of a H, a lower alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′ (wherein R′ is H or a        lower alkyl group);    -   R₄ and R₅ are the same or different and are each selected from        the group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,        halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄        alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl (including        C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl        (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl        (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄        alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato        (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO—),        carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄        alkyl)), arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl        (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN), isocyano        (—N⁺C—), cyanato (—O—CN), isocyanato (—O—N⁺═C—), isothiocyanato        (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl        (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino,        C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄        alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.),        alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,        alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where        R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso        (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O—), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato        (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations        thereof, and wherein R₄ and R₅ may be linked to form a cyclic or        polycyclic ring, wherein the ring is a substituted or        unsubstituted aryl, a substituted or unsubstituted heteroaryl, a        substituted or unsubstituted cycloalkyl, and a substituted or        unsubstituted heterocyclyl; and pharmaceutically acceptable        salts thereof.

In other embodiments, the 15-PGDH inhibitor can i) at 2.5 μMconcentration, stimulate a Vaco503 reporter cell line expressing a15-PGDH luciferase fusion construct to a luciferase output level ofgreater than 70 (using a scale on which a value of 100 indicates adoubling of reporter output over baseline); ii) at 2.5 μM concentrationstimulate a V9m reporter cell line expressing a 15-PGDH luciferasefusion construct to a luciferase output level of greater than 75; iii)at 7.5 μM concentration stimulate a LS174T reporter cell line expressinga 15-PGDH luciferase fusion construct to a luciferase output level ofgreater than 70; iv) at 7.5 μM concentration, does not activate anegative control V9m cell line expressing TK-renilla luciferase reporterto a level greater than 20; and v) inhibits the enzymatic activity ofrecombinant 15-PGDH protein at an IC50 of less than 1 μM.

In other embodiments, the 15-PGDH inhibitor can i) at 2.5 μMconcentration, stimulate a Vaco503 reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; ii)at 2.5 μM concentration stimulate a V9m reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iii)at 7.5 μM concentration stimulate a LS174T reporter cell line expressinga 15-PGDH luciferase fusion construct to increase luciferase output; iv)at 7.5 μM concentration, does not activate a negative control V9m cellline expressing TK-renilla luciferase reporter to a luciferase levelgreater than 20% above background; and v) inhibits the enzymaticactivity of recombinant 15-PGDH protein at an IC50 of less than 1 μM.

In other embodiments, the 15-PGDH inhibitor can inhibit the enzymaticactivity of recombinant 15-PGDH at an IC50 of less than 1 μM, orpreferably at an IC50 of less than 250 nM, or more preferably at an IC50of less than 50 nM, or more preferably at an IC50 of less than 5 nM.

In still other embodiments, the 15-PGDH inhibitor can be applied to skinof a subject to promote and/or stimulate pigmentation of the skin and/orhair growth and/or inhibit hair loss. The 15-PGDH inhibitor can also beadministered to a subject to promote wound healing, regenerate tissue,and/or treat at least one of oral ulcers, ulcerative colitis,gastrointestinal ulcers, inflammatory bowel disease, vascularinsufficiency, colitis, Raynaud's disease, Buerger's disease, diabeticneuropathy, pulmonary artery hypertension, cardiovascular disease,diabetic ulcers, renal disease, and erectile dysfunction. The 15-PGDHinhibitor can further be administered to a subject in combination with aprostanoid agonist for the purpose of enhancing the therapeutic effectof the agonist in prostaglandin responsive conditions.

In some embodiments, the 15-PGDH inhibitor can be administered to tissueof a subject to increase tissue stem cells. The 15-PGDH inhibitor canalso be administered to a bone marrow graft donor or a hematopoieticstem cell donor to increase the fitness of a donor bone marrow graft ora donor hematopoietic stem cell graft. The 15-PGDH inhibitor can beadministered to bone marrow of a subject to increase stem cells in thesubject. The 15-PGDH inhibitor can further be administered to bonemarrow of a subject to increase the fitness of the marrow as a donorgraft.

In other embodiments the 15-PGDH inhibitor can be administered to apreparation of hematopoietic stem cells of a subject to increase thefitness of the stem cell preparation as a donor graft. The 15-PGDHinhibitor can also be administered to a preparation of peripheral bloodhematopoietic stem cells of a subject to increase the fitness of thestem cell preparation as a donor graft. The 15-PGDH inhibitor canfurther be administered to a preparation of umbilical cord stem cells toincrease the fitness of the stem cell preparation as a donor graft.

In yet other embodiments, the 15-PGDH inhibitor can be administered to asubject to mitigate bone marrow graft rejection, to enhance bone marrowgraft engraftment, and/or to enhance engraftment of a hematopoietic stemcell graft, or an umbilical cord stem cell graft.

In still other embodiment, the 15-PGDH inhibitor can be administered toa subject or to a tissue graft of a subject to mitigate graft rejectionor to enhance graft engraftment.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject or to tissue of the subject to confer resistance to toxic orlethal effects of exposure to radiation.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject for the treatment of osteoporosis, bone fractures, or promotinghealing after bone injury or joint replacement.

In an alternative example, the 15-PGDH inhibitor can be administered toa subject or to the liver of a subject to promote liver regenerationfollowing liver resection or following toxic injury to the liver. In oneinstance, toxic injury to the liver may be caused by overdose ofacetaminophen or related hepatotoxic compounds.

In still other embodiments of the application, a 15-PGDH activator canbe administered to a tissue of a subject at an amount effective toincrease 15-PGDH levels and decrease prostaglandin levels in the tissue.The 15-PGDH activator can include formula (IV):

-   -   wherein X₃ and Y₂ are independently C or SO;    -   U is OR″(wherein R″ is H, a substituted or unsubstituted alkyl        group, or substituted or unsubstituted aryl group) or

-   -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each selected from the group        consisting of H, F, Cl, Br, I, an alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′        (wherein R′ is H or a lower alkyl group), a substituted or        unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a        substituted or unsubstituted heterocyclyl, and R₈ and R₉ may be        linked to form a cyclic or polycyclic ring; and pharmaceutically        acceptable salts thereof.

In some embodiments, the activator can i) at 7.5 μM concentration,stimulate a Vaco503 reporter cell line expressing a 15-PGDH luciferasefusion construct to a luciferase output level of greater than 50 (usinga scale on which a value of 100 indicates a doubling of reporter outputover baseline); ii) at 7.5 μM concentration stimulate a V9m reportercell line expressing a 15-PGDH luciferase fusion construct to aluciferase output level of greater than 50; iii) at 7.5 μM concentrationstimulate a LS174T reporter cell line expressing a 15-PGDH luciferasefusion construct to a luciferase output level of greater than 50; iv) at7.5 μM concentration, does not activate the negative control V9m cellline expressing TK-renilla luciferase reporter to a level any greaterthan 25; and v) against recombinant 15-PGDH protein the compound showsan IC₅₀ concentration for inhibiting 15-PGDH enzyme activity of greaterthan or equal to 2.5 μM.

In some embodiments, the activator can i) at 7.5 μM concentration,stimulate a Vaco503 reporter cell line expressing a 15-PGDH luciferasefusion construct to increase luciferase output; ii) at 7.5 μMconcentration stimulate a V9m reporter cell line expressing a 15-PGDHluciferase fusion construct to increase luciferase output; iii) at 7.5μM concentration stimulate a LS174T reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iv)at 7.5 μM concentration, does not activate the negative control V9m cellline expressing TK-renilla luciferase reporter to a luciferase level anygreater than 25% above; and v) against recombinant 15-PGDH protein thecompound shows an IC₅₀ concentration for inhibiting 15-PGDH enzymeactivity of greater than or equal to 2.5 μM.

In other embodiments, the 15-PGDH activator can be administered to asubject to treat a neoplasia, such as a colon neoplasia. The 15-PGDHactivator can also be administered to a subject to prevent neoplasia,such as a colon neoplasia. The 15-PGDH activator can also beadministered to a subject to reduce inflammation and/or pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows luciferase activity of Vaco-9m (V9m) cells that express a15-PGDH luciferase fusion construct created by targeted gene knock-in ofrenilla luciferase into the last coding exon of 15-PGDH treated with thecompounds SW033291, SW054384, and SW145753 at various concentrations.The activity is demonstrated in three different colon cancer cell linesall engineered to contain the 15-PGDH-luciferase fusion. These celllines are Vaco-9m (V9m), LS174T, Vaco503 (V503).

FIG. 1B shows luciferase activity of Vaco503 (V503) cells that express a15-PGDH luciferase fusion construct created by targeted gene knock-in ofrenilla luciferase into the last coding exon of 15-PGDH treated with thecompounds SW033291, SW054384, and SW145753 at various concentrations.

FIG. 1C shows luciferase activity of LS174T cells that express a 15-PGDHluciferase fusion construct created by targeted gene knock-in of renillaluciferase into the last coding exon of 15-PGDH treated with thecompounds SW033291, SW054384, and SW145753 at various concentrations.

FIG. 2 illustrates western blots demonstrating the levels of 15-PGDHprotein in cell lines V9M, LS174T, and V503 treated with 7.5 μM ofSW033291, SW054384, and SW145753 for 48 hours. Untreated FET cellsprovide a positive control for 15-PGDH expression.

FIG. 3 illustrates western blots demonstrating 15-PGDH protein levels incolon cell lines treated with SW124531 (FET cells treated with TGF-ß (10ng/ml for 48 hours) are used as a positive control for 15-PGDHexpression in certain panels).

FIG. 4 illustrates western blots demonstrating the levels of 15-PGDHprotein (wt-PGDH) expressed from a cDNA expression vector inV400-S3-2-32 cells treated with 5 μM SW124531, and protein levels of acatalytically dead mutant 15-PGDH (mu-PGDH) also expressed from a cDNAexpression vector in V400-M3-2-72 cells treated with SW124531.

FIG. 5A illustrates 15-PGDH protein levels in V503 cells treated withSW124531 as assayed by immuno-fluorescence.

FIG. 5B illustrates 15-PGDH protein levels in V503 cells treated withSW124531 by western blot.

FIG. 5C illustrates the chemical structure of SW124531.

FIG. 6A shows 15-PGDH mRNA levels in colon cancer cell line VM9 treatedwith SW033291.

FIG. 6B shows 15-PGDH mRNA levels in colon cancer cell line Ls174Ttreated with SW033291.

FIG. 6C shows 15-PGDH mRNA levels in colon cancer cell line V503 treatedwith SW033291.

FIG. 6D shows 15-PGDH mRNA levels in colon cancer cell line V9M-SC3treated with SW033291.

FIG. 6E illustrate graphs showing 15-PGDH mRNA levels in colon cancercell line Ls174T-SC1 treated with SW033291.

FIG. 6F illustrate graphs showing 15-PGDH mRNA levels in colon cancercell line V503-3H9-7 treated with SW033291.

FIG. 7A shows 15-PGDH mRNA levels in colon cancer cell line V503 treatedwith SW033291.

FIG. 7B shows 15-PGDH mRNA levels in colon cancer cell line LS174Ttreated with SW033291.

FIG. 7C shows 15-PGDH mRNA levels in colon cancer cell line V9M treatedwith SW033291.

FIG. 8A shows 15-PGDH mRNA levels in colon cancer cell line V503 treatedwith SW054384 and SW145753.

FIG. 8B shows 15-PGDH mRNA levels in colon cancer cell line Ls174Ttreated with SW054384 and SW145753.

FIG. 8C shows 15-PGDH mRNA levels in colon cancer cell line V9M treatedwith SW054384 and SW145753.

FIG. 9A shows 15-PGDH mRNA levels in colon cancer cell line FET treatedwith 5 μM SW124531.

FIG. 9B shows 15-PGDH mRNA levels in colon cancer cell line SW480treated with 5 μM SW124531.

FIG. 9C shows 15-PGDH mRNA levels in colon cancer cell line V503 treatedwith 5 μM SW124531.

FIG. 9D shows 15-PGDH mRNA levels in colon cancer cell line FET treatedwith 5 μM SW124531.

FIG. 9E shows 15-PGDH mRNA levels in colon cancer cell line SW480treated with 5 μM SW124531.

FIG. 9F shows 15-PGDH mRNA levels in colon cancer cell line V503 treatedwith 5 μM SW124531.

FIG. 9G shows 15-PGDH mRNA levels at 48 hours in colon cancer cell lineFET treated with 5 μM SW124531.

FIG. 9H shows 15-PGDH mRNA levels at 48 hours in colon cancer cell lineSW480 treated with 5 μM SW124531.

FIG. 9I shows 15-PGDH mRNA levels at 48 hours in colon cancer cell lineV503 treated with 5 μM SW124531.

FIG. 10A shows 15-PGDH activity in V503 treated with SW033291, SW054384,and SW145753. Activity is measured as pmol PGE₂/min/million cells.

FIG. 10B shows 15-PGDH activity in V9M treated with SW033291, SW054384,and SW145753. Activity is measured as pmol PGE₂/min/million cells.

FIG. 10C shows 15-PGDH activity in Ls174T treated with SW033291,SW054384, and SW145753. Activity is measured as pmol PGE₂/min/millioncells.

FIG. 11A illustrates a table and plot showing activity of recombinant15-PGDH protein (a 15-PGDH-GST fusion protein) incubated with varyingconcentrations of the test compounds.

FIG. 11B illustrates a plot showing activity of recombinant 15-PGDHprotein (a 15-PGDH-GST fusion protein) incubated with SW054384.

FIG. 11C illustrates a plot showing activity of recombinant 15-PGDHprotein (a 15-PGDH-GST fusion protein) incubated with Cayman.

FIG. 11D illustrates a plot showing activity of recombinant 15-PGDHprotein (a 15-PGDH-GST fusion protein) incubated with SW033291.

FIG. 12A shows the activity of recombinant 15-PGDH protein treated withSW033291 and SW054384, measuring transfer of tritium from a radiolabeledPGE₂ substrate.

FIG. 12B shows the activity of recombinant 15-PGDH protein treated withSW033291 and SW054384, measuring transfer of tritium from a radiolabeledPGE₂ substrate.

FIG. 12C shows the activity of recombinant 15-PGDH protein treated withSW033291 and SW054384, measuring generation of NADH by fluorescence.

FIG. 12D shows the activity of recombinant 15-PGDH protein treated withSW033291 and SW054384, measuring generation of NADH by fluorescence.

FIG. 13 illustrates a table and plot showing 15-PGDH activity measuredby following transfer of tritium from a radiolabeled PGE₂ substrate incells treated with SW124531 (upper panel) and in recombinant 15-PGDHprotein treated with SW124531 (lower panel).

FIG. 14A illustrate melt curves showing different compound's ability todirectly bind to recombinant 15-PGDH protein as measured by shifting themelting temperature of the protein.

FIG. 14B shows a table showing different compound's ability to directlybind to recombinant 15-PGDH protein as measured by shifting the meltingtemperature of the protein.

FIG. 15A illustrates a melt curve temperature of catalytically inactivemutant 15-PGDH protein treated with the test compounds.

FIG. 15B illustrates a melt curve temperature of catalytically inactivemutant 15-PGDH protein treated with the test compounds.

FIG. 16A illustrates a graph showing PGE₂ levels that are assayed in themedium of A549 cells that have been stimulated by IL1-beta for 23 hours,with the test compounds.

FIG. 16B illustrates a graph showing PGE₂ levels that are assayed in themedium of A549 cells that have been stimulated by IL1-beta for 23 hours,with the test compounds.

FIG. 17 illustrates a graph showing the dose response effect of SW033291on PGE₂ production from IL1-beta treated A549 cells.

FIG. 18A illustrates a graph showing the in vivo modulations bycompounds (2.5 μM) of PGDH activity as reflected in PGE₂ levelsfollowing addition of PGE₂ into the medium of Vaco-503 cells.

FIG. 18B illustrates a graph showing the in vivo normalized modulationsby compounds (2.5 μM) of PGDH activity as reflected in PGE₂ levelsfollowing addition of PGE₂ into the medium of Vaco-503 cells.

FIG. 19 illustrates images showing the activity of SW033291 in speedingthe healing of a model wound consisting of a scratch in a monolayer ofHaCaT cells observed over 48 hours of treatment.

FIG. 20A illustrates a graph showing the quantitation of scratch widthat 0 and 48 hours in the control, SW033291 (2.5 μM) treated cells, andthe TGF-beta (1 ng/ml) treated cells.

FIG. 20B illustrates a graph showing the quantitation of scratch widthat 48 hours, SW033291 (2.5 μM) treated cells, and the TGF-beta (1 ng/ml)treated cells.

FIG. 21A shows percent inhibition of PGDH using titrations of 15-PGDHinhibitor SW033291 run at different 15-PGDH concentrations.

FIG. 21B shows the IC50 of 15-PGDH inhibitor SW033291 versus 15-PGDHconcentration.

FIG. 22A shows 15-PGDH enzyme inhibiting activity.

FIG. 22B shows percent inhibition of activity due to of SW033291 asmeasured before and after dialysis of the 15-PGDH and SW033291 mixture.

FIG. 23A illustrates a plot showing reaction rates of 15-PGDH at varyingconcentrations of SW033291.

FIG. 23B illustrates a plot showing relative reaction velocity of15-PGDH at varying concentrations of SW033291.

FIG. 24A illustrate a plot showing inhibition of 15-PGDH by SW033291 inthe presence of PGE-2.

FIG. 24B illustrate a plot showing IC50 of SW033291 against 15-PGDHversus PGE₂ concentration.

FIG. 25 illustrates a schematic diagram showing the structure activityrelationships of analogues of SW033291 versus their IC50 against15-PGDH.

FIG. 26 illustrates a schematic diagram showing additional analogues ofSW033291.

FIG. 27A illustrates a graph showing luciferase activity of colon cancercell line V9M-SC3 treated with 2.5 μM and 7.5 μM the compounds of FIG.26 .

FIG. 27B illustrates graph showing luciferase activity of colon cancercell line V503 treated with 2.5 μM and 7.5 μM the compounds of FIG. 26 .

FIG. 27C illustrates a graph showing luciferase activity of colon cancercell line Ls174T treated with 2.5 μM and 7.5 μM the compounds of FIG. 26.

FIG. 28 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 26 .

FIG. 29A illustrates a plot showing the IC50 against 15-PGDH of SW033291and SW0206980.

FIG. 29B illustrates a plot showing the IC50 against 15-PGDH of SW033291and SW0206980.

FIG. 30A illustrates a plot showing melting profiles of SW0206890 andSW033291 binding to 15-PGDH.

FIG. 30B illustrates a plot showing melting profiles of SW0206890 andSW033291 binding to 15-PGDH.

FIG. 31A illustrates a plot showing percent inhibition of 15-PGDHactivity by SW033291, SW206980, and SW206992.

FIG. 31B illustrates a plot showing percent inhibition of 15-PGDHactivity by SW033291, SW206980, and SW206992.

FIG. 31C illustrates a plot showing percent inhibition of 15-PGDHactivity by SW033291, SW206980, and SW206992.

FIG. 32A illustrates a graph showing luciferase activity of colon cancercell line V9m-SC3 treated with various concentrations of SW033291.

FIG. 32B illustrates a graph showing luciferase activity of colon cancercell line Ls174T treated with various concentrations of SW033291.

FIG. 32C illustrates a graph showing luciferase activity of colon cancercell line V503 treated with various concentrations of SW033291.

FIG. 33A illustrates a graph showing luciferase activity of colon cancercell line V9M-SC3 treated with various concentrations of SW0206980.

FIG. 33B illustrates a graph showing luciferase activity of colon cancercell line LS174T treated with various concentrations of SW0206980.

FIG. 33C illustrates a graph showing luciferase activity of colon cancercell line V503 treated with various concentrations of SW0206980.

FIG. 34A illustrates a graph showing luciferase activity of colon cancercell line V9M-SC3 treated with various concentrations of SW0206992.

FIG. 34B illustrates a graph showing luciferase activity of colon cancercell line LS174T treated with various concentrations of SW0206992.

FIG. 34C illustrates a graph showing luciferase activity of colon cancercell line V503 treated with various concentrations of SW0206992.

FIG. 35A illustrates a plot showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIG. 35B illustrates a plot showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIG. 36A illustrates a plot showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIG. 36B illustrates a plot showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIG. 37A illustrates a graph showing the effect of SW033291 on theregulation of PGE-2 in A549 cells stimulated with IL1-Beta.

FIG. 37B illustrates a graph showing the effect of SW0206890 on theregulation of PGE-2 in A549 cells stimulated with IL1-Beta.

FIG. 37C illustrates a graph showing the effect of SW206992 on theregulation of PGE-2 in A549 cells stimulated with IL1-Beta.

FIG. 38A illustrates a graph showing the effect of SW033291 on cellnumbers in A549 cells after stimulated with IL1-Beta.

FIG. 38B illustrates a graph showing the effect of SW0206890 on cellnumbers in A549 cells after stimulated with IL1-Beta.

FIG. 38C illustrates a graph showing the effect of SW206992 on cellnumbers in A549 cells after stimulated with IL1-Beta.

FIG. 39 illustrates a schematic diagram of additional analogues ofSW033291.

FIG. 40A illustrates a graph showing luciferase activity of colon cancercell line V9M, LS174T, and V503 treated with 2.5 μM and 7.5 μM thecompounds of FIG. 39 .

FIG. 40B illustrates a graph showing luciferase activity of colon cancercell line LS174T treated with 2.5 μM and 7.5 μM the compounds of FIG. 39.

FIG. 40C illustrates a graph showing luciferase activity of colon cancercell line V503 treated with 2.5 μM and 7.5 μM the compounds of FIG. 39 .

FIG. 41 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 40 .

FIG. 42A illustrates a graph showing percent inhibition of 15-PGDHactivity by SW208064.

FIG. 42B illustrates a graph showing percent inhibition of 15-PGDHactivity by SW208065.

FIG. 42C illustrates a graph showing percent inhibition of 15-PGDHactivity by SW208066.

FIG. 42D illustrates a graph showing percent inhibition of 15-PGDHactivity by SW208067.

FIG. 43 shows the dose response curve for induction of a15-PGDH-luciferase fusion gene reporter in the V9m cell line backgroundof SW033291, SW208064, SW208065, SW208066, and SW208067.

FIG. 44 illustrates titration curves of 15-PGDH inhibitor compounds inan assay measuring effects on PGE₂ levels in the medium of A549 cellsthat have been stimulated with IL1-beta.

FIG. 45 is a plot showing weight change of FVB mice treated withSW033291.

FIG. 46A shows total bone marrow cellularity.

FIG. 46B shows SKL population of wild type versus PGDH−/− mice.

FIG. 46C shows average CFU counts in wild type versus PGDH−/− mice(designated as either PGDH −/− or as PGDH).

FIG. 47 illustrates a graph showing CFU counts in wild type bone marrowtreated with SW033291 and PGE-2.

FIG. 48A illustrates a graph showing bone marrow cellularity of micetreated with SW033291.

FIG. 48B illustrates a graph showing SKL % in whole bone marrow of micetreated with SW033291.

FIG. 48C illustrates a graph showing CFU counts in mice treatedSW033291.

FIG. 49A shows a schematic diagram following CD45.2 antigen marked cellsin lethally irradiated C57BL/6J mice rescued with a bone marrowtransplant from donor mice treated with SW033291 or with vehicle.

FIG. 49B shows graphs showing chimerism, of donor B-Cells, myeloidcells, and T-Cells after such treatment.

FIG. 50 illustrates a schematic diagram showing schema of a study inwhich C57BL/61 mice are irradiated with 11GY on day 0 and followed bytreatment with SW033291.

FIG. 51 illustrates a schematic diagram of a partial hepatectomy.

FIG. 52A shows photographs of preoperative view of mouse liver.

FIG. 52B shows photographs of preoperative view of mouse liver.

FIG. 52C shows photographs of post-operative view of mouse liver.

FIG. 52D shows photographs of post-operative view of mouse liver.

FIG. 53A shows a photograph of post-hepatectomy views of the mouse liver(at left).

FIG. 53B shows a photograph of regeneration of mouse liver onpost-operative day 7 (at right).

FIG. 53C shows a photograph of post-hepatectomy views of the mouse liver(at left).

FIG. 53D show a photograph of regeneration of mouse liver onpost-operative day 7 (at right).

FIG. 54A shows a micrograph of post-hepatectomy mouse livers of mouseadministered SW033291, with arrows designating mitotic figures.

FIG. 54B shows a micrograph of post-hepatectomy mouse livers of mouseadministered control vehicle, with arrows designating mitotic figures.

FIG. 55 illustrates a graph showing mitosis in liver of SW033291 treatedmouse versus the control mouse.

FIG. 56 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated C57B1/6J mice.

FIG. 57 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291 twicedaily treated C57B1/6J mice.

FIG. 58 illustrates a graph reprising the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated C57B1/6J mice.

FIG. 59A illustrates a graph showing ALT levels following partialhepatectomy in one mouse control versus one mouse treated with SW033291.

FIG. 59B illustrates a plot showing ALT levels following partialhepatectomy in one mouse control versus one mouse treated with SW033291.

FIG. 60 illustrates a graph showing serum bilirubin levels followingpartial hepatectomy in a control mouse and a mouse treated withSW033291.

FIG. 61 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated FVB mice.

FIG. 62 illustrates a graph showing preoperative body weights in controlversus SW033291 treated FVB mice.

FIG. 63 illustrates a graph showing the weight of the resected liversegment from mice treated with either SW033291 or vehicle control andassayed for liver regeneration.

FIG. 64 illustrates a graph showing liver weights attained post partialheptatectomy in SW033291 and control mice.

FIG. 65 illustrates a graph showing the liver to body weight ratiosobtained post partial hepatectomy in SW033291 treated and control mice.

FIG. 66 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 4.

FIG. 67 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 7.

FIG. 68 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 4.

FIG. 69 illustrates photographs of S-phase cells following partialhepatectomy on post-operative day 2 in livers of SW033291 treated andvehicle treated control mice.

FIG. 70 illustrates a photograph showing high powered (40×) views ofrepresentative fields from the study of FIG. 69 .

FIG. 71 illustrates a “box and whiskers” plot comparing percent of BrdUpositive cells in livers of SW033291 treated versus vehicle controltreated mice on post-operative day 2 following partial hepatectomy.

FIG. 72 illustrates a graph showing the average changes from baselineweight of the cohort of control versus SW033291 treated mice all treatedwith 2% dextran sulfate sodium (DSS) in the drinking water.

FIG. 73 illustrates a graph of the daily disease activity index of thecohort of control versus SW033291 treated mice all treated with 2% DSSin the drinking water.

FIG. 74 illustrates a graph showing the average changes from baselineweight of the cohort of DSS treated mice receiving a control vehicleversus SW033291.

FIG. 75A shows a graph of the number of ulcers in a colon of DSS treatedmice receiving a control vehicle versus SW033291.

FIG. 75B shows photographs of ulcers of DSS treated mice receivingcontrol (left) or SW033291 (right).

FIG. 76 illustrates a graph showing quantitation of ulcer burden on day15 of DSS treated mice.

FIG. 77A shows a photograph of colonoscopic findings and mouseendoscopic index of colitis severity (MEICs) for a DSS treated mousereceiving SW033291.

FIG. 77B shows a photograph of colonoscopic findings and mouseendoscopic index of colitis severity (MEICs) for a DSS treated mousereceiving a control vehicle.

FIG. 78 illustrates a graph showing MEICS score of DSS treated micereceiving a control vehicle or SW033291.

FIG. 79 illustrates photomicrographs of high powered fields from themid-colon on day 8 of the DSS protocol from control mice, SW033291treated mice (treatment) and 15-PGDH knockout mice (KO) and a graphdepicting sum of the average number of BrdU positive cells per crypt inthe distal plus middle colons of control (Cn), SW033219 treated mice(Tx), and 15-PGDH knockout mice (KO) on day 1, day 8, and day 15 of theDSS treatment protocol.

FIG. 80 illustrates a graph showing colon length at day 22 of DSStreated mice receiving a control vehicle or SW033291.

FIG. 81 illustrates a schematic diagram of analogues of SW054384.

FIG. 82A shows luciferase activity of colon cancer cell line V9M treatedwith 2.5 μM and 7.5 μM the compounds of FIG. 81 .

FIG. 82B shows luciferase activity of colon cancer cell line V503treated with 2.5 μM and 7.5 μM the compounds of FIG. 81 .

FIG. 82C shows luciferase activity of colon cancer cell line LS174Ttreated with 2.5 μM and 7.5 μM the compounds of FIG. 81 .

FIG. 83 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 74 .

FIG. 84A shows a graph showing activity in lowering PGE₂ levels in mediaof A549 cells that are stimulated to produce PEG2 by treatment usingIL1-beta using compounds of FIG. 81 .

FIG. 84B shows a graph showing toxicity of A549 cells administered thecompounds of FIG. 81 .

FIG. 84C shows a photographs of A549 cells treated with compounds ofFIG. 81 .

FIG. 85 illustrates a plot showing metabolic stability of SW054384 byincubation with murine liver S9 microsomes.

FIG. 86 illustrates a plot showing metabolic stability of SW0125991 byincubation with murine liver S9 microsomes.

FIG. 87 illustrates a schematic diagram of analogues of SW054384.

FIG. 88 illustrates a graph showing luciferase activity of colon cancercell V9m treated with 2.5 μM and 7.5 μM the compounds of FIG. 87 .

FIG. 89 illustrates a graph showing luciferase activity of colon cancerLS174T cells treated with 2.5 μM and 7.5 μM the compounds of FIG. 87 .

FIG. 90 illustrates a graph showing luciferase activity of colon cancercell V503 treated with 2.5 μM and 7.5 μM the compounds of FIG. 87 .

FIG. 91 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 87 .

FIG. 92 is a schematic illustration showing the structures of 15-PGDHactivators SW054384, SW125991, SW207997, SW207998, and SW207999.

FIG. 93 illustrates a graph showing the activities of SW054384,SW125991, SW207997, SW207998, SW207999 in lowering PGE₂ levels in mediumof A549 cells that have been treated with 2.5 μM of each compound alongwith addition of 2.5 ng/ml IL1-beta.

FIG. 94 illustrates titration curves of 15-PGDH activator compounds inan assay measuring effects on PGE₂ levels in the medium of A549 cellsthat have been stimulated with IL1-beta.

FIG. 95 illustrates a photograph showing assessment of toxicity ofSW125991 by testing effect of increasing doses on colony formation ofA549 cells, Vaco9M (V9m) cells, LS174T cells, and Vaco503 (V503) cells.

FIG. 96 illustrates a photograph showing assessment of toxicity ofSW207997 by testing effect of increasing doses on colony formation ofA549 cells, Vaco9M (V9m) cells, LS174T cells, and Vaco503 (V503) cells.

FIG. 97 illustrates a photograph showing assessment of toxicity ofSW207998 by testing effect of increasing doses on colony formation ofA549 cells, Vaco9M (V9m) cells, LS174T cells, and Vaco503 (V503) cells.

FIG. 98 illustrates a photograph showing assessment of toxicity ofSW207999 by testing effect of increasing doses on colony formation ofA549 cells, Vaco9M (V9m) cells, LS174T cells, and Vaco503 (V503) cells.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

It will be noted that the structure of some of the compounds of theapplication include asymmetric (chiral) carbon atoms. It is to beunderstood accordingly that the isomers arising from such asymmetry areincluded herein, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. The compounds of thisapplication may exist in stereoisomeric form, therefore can be producedas individual stereoisomers or as mixtures.

The term “isomerism” means compounds that have identical molecularformulae but that differ in the nature or the sequence of bonding oftheir atoms or in the arrangement of their atoms in space. Isomers thatdiffer in the arrangement of their atoms in space are termed“stereoisomers”. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers”, and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four nonidentical substituentsis termed a “chiral center”.

The term “chiral isomer” means a compound with at least one chiralcenter. It has two enantiomeric forms of opposite chirality and mayexist either as an individual enantiomer or as a mixture of enantiomers.A mixture containing equal amounts of individual enantiomeric forms ofopposite chirality is termed a “racemic mixture”. A compound that hasmore than one chiral center has 2n−1 enantiomeric pairs, where n is thenumber of chiral centers. Compounds with more than one chiral center mayexist as either an individual diastereomer or as a mixture ofdiastereomers, termed a “diastereomeric mixture”. When one chiral centeris present, a stereoisomer may be characterized by the absoluteconfiguration (R or S) of that chiral center. Alternatively, when one ormore chiral centers are present, a stereoisomer may be characterized as(+) or (−). Absolute configuration refers to the arrangement in space ofthe substituents attached to the chiral center. The substituentsattached to the chiral center under consideration are ranked inaccordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn etal, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London),612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,41, 116).

The term “geometric Isomers” means the diastereomers that owe theirexistence to hindered rotation about double bonds. These configurationsare differentiated in their names by the prefixes cis and trans, or Zand E, which indicate that the groups are on the same or opposite sideof the double bond in the molecule according to the Cahn-Ingold-Prelogrules. Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof.

The term “atropic isomers” are a type of stereoisomer in which the atomsof two isomers are arranged differently in space. Atropic isomers owetheir existence to a restricted rotation caused by hindrance of rotationof large groups about a central bond. Such atropic isomers typicallyexist as a mixture, however as a result of recent advances inchromatography techniques, it has been possible to separate mixtures oftwo atropic isomers in select cases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “treating” is art-recognized and includes inhibiting a disease,disorder or condition in a subject, e.g., impeding its progress; andrelieving the disease, disorder or condition, e.g., causing regressionof the disease, disorder and/or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected.

The term “preventing” is art-recognized and includes stopping a disease,disorder or condition from occurring in a subject, which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds of the application are capable of further forming salts.All of these forms are also contemplated herein.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. For example, the saltcan be an acid addition salt. One embodiment of an acid addition salt isa hydrochloride salt. The pharmaceutically acceptable salts can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrilebeing preferred. Lists of salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990).

The compounds described herein can also be prepared as esters, forexample pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, ester groups (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl)N-Mannich bases, Schiff bases and enaminonesof amino functional groups, oximes, acetals, ketals and enol esters ofketone and aldehyde functional groups in compounds of Formula I, and thelike, See Bundegaard, H. “Design of Prodrugs” p1-92, Elesevier, NewYork-Oxford (1985).

The term “protecting group” refers to a grouping of atoms that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Examples of protecting groups can be found in Green andWuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed.1991); Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski,Protecting Groups, (Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Those of skill in the art can identify othersuitable amine protecting groups.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

Additionally, the salts of the compounds described herein, can exist ineither hydrated or unhydrated (the anhydrous) form or as solvates withother solvent molecules. Nonlimiting examples of hydrates includemonohydrates, dihydrates, etc. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The term “solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The compounds, salts and prodrugs described herein can exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. Tautomers existas mixtures of a tautomeric set in solution. In solid form, usually onetautomer predominates. Even though one tautomer may be described, thepresent application includes all tautomers of the present compounds. Atautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs.

Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2.formation of a delocalized anion (e.g., an enolate); 3. protonation at adifferent position of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

The term “analogue” refers to a chemical compound that is structurallysimilar to another but differs slightly in composition (as in thereplacement of one atom by an atom of a different element or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analogue is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. A patient refers to a subjectafflicted with a disease or disorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. Incertain embodiments, a therapeutically effective amount of a therapeuticagent for in vivo use will likely depend on a number of factors,including: the rate of release of an agent from a polymer matrix, whichwill depend in part on the chemical and physical characteristics of thepolymer; the identity of the agent; the mode and method ofadministration; and any other materials incorporated in the polymermatrix in addition to the agent.

The term “ED50” is art-recognized. In certain embodiments, ED50 meansthe dose of a drug, which produces 50% of its maximum response oreffect, or alternatively, the dose, which produces a pre-determinedresponse in 50% of test subjects or preparations. The term “LD50” isart-recognized. In certain embodiments, LD50 means the dose of a drug,which is lethal in 50% of test subjects. The term “therapeutic index” isan art-recognized term, which refers to the therapeutic index of a drug,defined as LD50/ED50.

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc.

With respect to any chemical compounds, the present application isintended to include all isotopes of atoms occurring in the presentcompounds. Isotopes include those atoms having the same atomic numberbut different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include C-13 and C-14.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent can be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent can be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When an atom or a chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it is meant to encompass each number within therange as well as all intermediate ranges. For example, “C₁₋₆ alkyl” ismeant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3,1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” is intended to include both branched (e.g., isopropyl,tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl(e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groupshave a specified number of carbon atoms. For example, C₁₋₆ alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As usedherein, “lower alkyl” refers to alkyl groups having from 1 to 6 carbonatoms in the backbone of the carbon chain. “Alkyl” further includesalkyl groups that have oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more hydrocarbon backbone carbon atoms. In certainembodiments, a straight chain or branched chain alkyl has six or fewercarbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ forbranched chain), for example four or fewer. Likewise, certaincycloalkyls have from three to eight carbon atoms in their ringstructure, such as five or six carbons in the ring structure.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). If not otherwise indicated, the terms “alkyl” and “loweralkyl” include linear, branched, cyclic, unsubstituted, substituted,and/or heteroatom-containing alkyl or lower alkyl, respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The terms “alkyl”, “alkenyl”, and “alkynyl” are intended to includemoieties which are diradicals, i.e., having two points of attachment. Anonlimiting example of such an alkyl moiety that is a diradical is—CH₂CH₂—, i.e., a C₂ alkyl group that is covalently bonded via eachterminal carbon atom to the remainder of the molecule.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Examples of arylgroups include benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diaryl amino, and al kylaryl amino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). If nototherwise indicated, the term “aryl” includes unsubstituted,substituted, and/or heteroatom-containing aromatic substituents.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The terms “heterocyclyl” or “heterocyclic group” include closed ringstructures, e.g., 3- to 10-, or 4- to 7-membered rings, which includeone or more heteroatoms. “Heteroatom” includes atoms of any elementother than carbon or hydrogen. Examples of heteroatoms include nitrogen,oxygen, sulfur and phosphorus.

Heterocyclyl groups can be saturated or unsaturated and includepyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine,lactones, lactams, such as azetidinones and pyrrolidinones, sultams, andsultones. Heterocyclic groups such as pyrrole and furan can havearomatic character. They include fused ring structures, such asquinoline and isoquinoline. Other examples of heterocyclic groupsinclude pyridine and purine. The heterocyclic ring can be substituted atone or more positions with such substituents as described above, as forexample, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety.Heterocyclic groups can also be substituted at one or more constituentatoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy,a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, or —CN, or the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.“Counterion” is used to represent a small, negatively charged speciessuch as fluoride, chloride, bromide, iodide, hydroxide, acetate, andsulfate.

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O—), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation, and asappropriate, purification from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The terms “free compound” is used herein to describe a compound in theunbound state.

Throughout the description, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “small molecule” is an art-recognized term. In certainembodiments, this term refers to a molecule, which has a molecularweight of less than about 2000 amu, or less than about 1000 amu, andeven less than about 500 amu.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

The term “neoplasm” refers to any abnormal mass of cells or tissue as aresult of neoplasia. The neoplasm may be benign, potentially malignant(precancerous), or malignant (cancerous). An adenoma is an example of aneoplasm.

The terms “adenoma”, “colon adenoma” and “polyp” are used herein todescribe any precancerous neoplasm of the colon.

The term “colon” as used herein is intended to encompass the right colon(including the cecum), the transverse colon, the left colon and therectum.

The terms “colorectal cancer” and “colon cancer” are usedinterchangeably herein to refer to any cancerous neoplasia of the colon(including the rectum, as defined above).

The terms “gene expression” or “protein expression” includes anyinformation pertaining to the amount of gene transcript or proteinpresent in a sample, as well as information about the rate at whichgenes or proteins are produced or are accumulating or being degraded(e.g., reporter gene data, data from nuclear runoff experiments,pulse-chase data etc.). Certain kinds of data might be viewed asrelating to both gene and protein expression. For example, proteinlevels in a cell are reflective of the level of protein as well as thelevel of transcription, and such data is intended to be included by thephrase “gene or protein expression information”. Such information may begiven in the form of amounts per cell, amounts relative to a controlgene or protein, in unitless measures, etc.; the term “information” isnot to be limited to any particular means of representation and isintended to mean any representation that provides relevant information.The term “expression levels” refers to a quantity reflected in orderivable from the gene or protein expression data, whether the data isdirected to gene transcript accumulation or protein accumulation orprotein synthesis rates, etc.

The terms “healthy” and “normal” are used interchangeably herein torefer to a subject or particular cell or tissue that is devoid (at leastto the limit of detection) of a disease condition.

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include analogues of eitherRNA or DNA made from nucleotide analogues, and, as applicable to theembodiment being described, single-stranded (such as sense or antisense)and double-stranded polynucleotides. In some embodiments, “nucleic acid”refers to inhibitory nucleic acids. Some categories of inhibitorynucleic acid compounds include antisense nucleic acids, RNAi constructs,and catalytic nucleic acid constructs. Such categories of nucleic acidsare well-known in the art.

Embodiments described herein relate to compounds and methods ofmodulating 15-PGDH activity, modulating tissue prostaglandin levels,and/or treating diseases, disorders, or conditions in which it isdesired to modulate 15-PGDH activity and/or prostaglandin levels.“Inhibitors,” “activators,” and “modulators” of 15-PGDH expression or of15-PGDH activity are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for 15-PGDH expression or 15-PGDH activity, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression of 15-PGDH or bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate the activity of 15-PGDH, e.g.,antagonists. Activators are agents that, e.g., induce or activate theexpression of a 15-PGDH or bind to, stimulate, stabilize, increase,open, activate, facilitate, or enhance activation, sensitize or upregulate the activity of 15-PGDH, e.g., agonists. Modulators includenaturally occurring and synthetic ligands, small chemical molecules, andthe like.

15-PGDH inhibitors described herein can provide a pharmacologic methodfor elevating prostaglandin levels in tissue. Known activities ofprostaglandins include promoting hair growth, promoting skinpigmentation, and promoting skin darkening or the appearance of skintanning. Known activities of prostaglandins also include amelioratingpulmonary artery hypertension. 15-PGDH inhibitors described herein mayalso be utilized to increase tissue stem cell numbers for purposes thatwould include increasing resistance to tissue damage by radiation,increasing resistance to environmental exposures to radiation,increasing stem cell numbers to increase fitness of bone marrow or othertypes of transplantation (through either in vivo exposure to 15-PGDHinhibitors described herein to increase stem cell numbers prior toharvest of a transplanted tissue, or through ex vivo exposure of aharvested tissue prior to transplant into a recipient host). 15-PGDHinhibitors described herein may also be utilized for purposes that wouldinclude promoting liver regeneration, including liver regeneration afterliver resection, and liver regeneration after toxic insults, which forexample may be the toxic insult of acetaminophen overdose. Prostaglandinsignaling is also known to promote wound healing, protect the stomachfrom ulceration, and promote healing of ulcers of stomach andintestines. Additionally, 15-PGDH inhibitors described herein canpromote activity of human keratinocytes in “healing” scratches acrosscultures of keratinocyte cells. Hence, 15-PGDH inhibitors describedherein may be utilized to also heal ulcers of other tissues, including,but not limited to skin, and including but not limited to diabeticulcers. Further, 15-PGDH inhibitors described herein may be utilized forthe treatment of erectile dysfunction.

15-PGDH activators described herein can increase levels of 15-PGDHprotein in cells and in increase levels of 15-PGDH enzymatic activity incells. Increasing tissue levels of 15-PGDH can decrease tissue levels ofprostaglandins. Activities associated with compounds that decreasetissue prostaglandins include decreasing development of human tumors,particularly decreasing development of human colon tumors. Accordingly,compounds that increase tissue 15-PGDH activity can lower risk ofdevelopment of colon and other tumors. Compounds that increase 15-PDGHactivity can also be used to treat colon and other tumors. Compoundsthat increase 15-PDGH may be used to treat or to prevent tumors whengiven singly, or when given in combination with inhibitors ofcyclooxygenase-1 and/or cyclooxygenase-2 enzymes, or when given incombination with other therapeutic agents.

15-PGDH inhibitors and activators described herein can be identifiedusing assays in which putative modulator compounds are applied to cellsexpressing 15-PGDH and then the functional effects on 15-PGDH activityare determined. Samples or assays comprising 15-PGDH that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of effect. Control samples (untreated withmodulators) are assigned a relative 15-PGDH activity value of 100%.Inhibition of 15-PGDH is achieved when the 15-PGDH activity valuerelative to the control is about 80%, optionally 50% or 25%, 10%, 5% or1%. Activation of 15-PGDH is achieved when the 15-PGDH activity orexpression value relative to the control is 105%, optionally 110%,optionally 125%, optionally 150%, optionally 200%, 300%, 400%, 500%, or1000-3000% or more higher.

Agents tested as modulators of 15-PGDH can be any small chemicalmolecule or compound. Typically, test compounds will be small chemicalmolecules, natural products, or peptides. The assays are designed toscreen large chemical libraries by automating the assay steps andproviding compounds from any convenient source to assays, which aretypically run in parallel (e.g., in microtiter formats on microtiterplates in robotic assays). Modulators also include agents designed toincrease the level of 15-PGDH mRNA or the level of translation from anmRNA.

In some embodiments, the modulator of 15-PGDH can be a 15-PGDH inhibitorthat includes a compound having the following formula (I):

-   -   wherein n is 0-2;    -   R₁ is a C₁₋₈ alkyl, which is linear, branched, or cyclic and        which is unsubstituted or substituted (e.g., R₁ can be C₂₋₆        alkyl, C₂₋₄ alkyl, or C₄ alkyl, which is linear, branched, or        cyclic and which is unsubstituted or substituted); R₂ and R₃ are        the same or different and are each selected from the group        consisting of a H, a lower alkyl group, (CH₂)_(n1)OR′ (wherein        n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X,        O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN, (C═O)—R′,        (C═O)N(R′)₂, O(CO)R′, COOR′ (wherein R′ is H or a lower alkyl        group);    -   R₄ and R₅ are the same or different and are each selected from        the group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,        halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄        alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl (including        C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl        (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl        (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄        alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato        (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO—),        carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄        alkyl)), arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl        (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN), isocyano        (—N⁺C—), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato        (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl        (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino,        C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄        alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.),        alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,        alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where        R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso        (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O—), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato        (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations        thereof, and wherein R₄ and R₅ may be linked to form a cyclic or        polycyclic ring, wherein the ring is a substituted or        unsubstituted aryl, a substituted or unsubstituted heteroaryl, a        substituted or unsubstituted cycloalkyl, and a substituted or        unsubstituted heterocyclyl; and pharmaceutically acceptable        salts thereof.

In other embodiments, the 15-PGDH inhibitor can include a compoundhaving the following formula (II):

-   -   wherein n is 0-2;    -   R₁ is a C₁₋₈ alkyl, which is linear, branched, or cyclic, and        which is unsubstituted or substituted;    -   R₂ and R₃ are the same or different and are each selected from        the group consisting of a H, a lower alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′ (wherein R′ is H or a        lower alkyl group);    -   Z₁ is NR′, O or S (wherein R′ is H or a lower alkyl group);    -   X₁ and Y₁ are the same or different and are each selected from        the group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,        halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄        alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl (including        C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl        (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl        (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄        alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato        (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO⁻),        carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄        alkyl)), arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl        (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN), isocyano        (—N⁺C—), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato        (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl        (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino,        C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄        alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.),        alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,        alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where        R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso        (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato        (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations        thereof, and wherein X₁ and Y₁ may be linked to form a cyclic or        polycyclic ring, wherein the ring is a substituted or        unsubstituted aryl, a substituted or unsubstituted heteroaryl, a        substituted or unsubstituted cycloalkyl, and a substituted or        unsubstituted heterocyclyl; and pharmaceutically acceptable        salts thereof.

Examples of 15-PGDH inhibitors having formulas (I) or (II) include thefollowing compounds:

and pharmaceutically acceptable salts thereof.

In certain embodiments, the 15-PGDH inhibitor having formula (I) or (II)can be selected that can ia) at 2.5 μM concentration, stimulate aVaco503 reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 70 (using a scaleon which a value of 100 indicates a doubling of reporter output overbaseline); iia) at 2.5 μM concentration stimulate a V9m reporter cellline expressing a 15-PGDH luciferase fusion construct to a luciferaseoutput level of greater than 75; iiia) at 7.5 μM concentration stimulatea LS174T reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 70; and iva) at7.5 μM concentration, does not activate a negative control V9m cell lineexpressing TK-renilla luciferase reporter to a level greater than 20;and va) inhibits the enzymatic activity of recombinant 15-PGDH proteinat an IC50 of less than 1 μM

In other embodiments, the 15-PGDH inhibitor can ib) at 2.5 μMconcentration, stimulate a Vaco503 reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iib)at 2.5 μM concentration stimulate a V9m reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iiib)at 7.5 μM concentration stimulate a LS174T reporter cell line expressinga 15-PGDH luciferase fusion construct to increase luciferase output;ivb) at 7.5 μM concentration, does not activate a negative control V9mcell line expressing TK-renilla luciferase reporter to a luciferaselevel greater than 20% above background; and vb) inhibits the enzymaticactivity of recombinant 15-PGDH protein at an IC50 of less than 1 μM.

In other embodiments, the 15-PGDH inhibitor can inhibit the enzymaticactivity of recombinant 15-PGDH at: ic) an IC50 of less than luM, orpreferably iic) at an IC50 of less than 250 nM, or more preferably iiic)at an IC50 of less than 50 nM, or more preferably iv) at an IC50 of lessthan 5 nM.

An example of a 15-PGDH inhibitor having formula (I) that meets theabove noted criteria (ia-va) includes a compound having the formula(III): An example of a 15-PGDH inhibitor having formula (I) that meetsthe above noted criteria (ib-vb) includes a compound having the formula(III): An example of a 15-PGDH inhibitor having formula (I) that meetsthe above noted criteria ic, and/or iic, and or iiic, and or ivc,includes a compound having the formula (III): In still otherembodiments, the 15-PGDH inhibitor can include a compound having thefollowing formula (III):

-   -   wherein n is 0-2;    -   R₁ is a C₁₋₈ alkyl, which is linear, branched, or cyclic and        which is unsubstituted or substituted;    -   R₂ and R₃ are the same or different and are each selected from        the group consisting of a H, a lower alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′ (wherein R′ is H or a        lower alkyl group);    -   Z₁ is NR′, O or S (wherein R′ is H or a lower alkyl group);    -   X₂ is N or C;    -   R₆ and R₇ are optional and if present are the same or different        and are each selected from the group consisting of a H, F, Cl,        Br, I, a lower alkyl group, (CH₂)_(n1)OR′ (wherein n1=1, 2, or        3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein        X═F, Cl, Br, or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,        O(CO)R′, OR′, SR′, COOR′ (wherein R′ is H or a lower alkyl        group); substituted or unsubstituted aryl, a substituted or        unsubstituted cycloalkyl, and a substituted or unsubstituted        heterocyclyl; and pharmaceutically acceptable salts thereof.

15-PGDH inhibitors having formula (III) can be synthesized as shown:

Any reaction solvent can be used in the above preparation process aslong as it is not involved in the reaction. For example, the reactionsolvent includes ethers such as diethyl ether, tetrahydrofuran anddioxane; halogenized hydrocarbons, such as dichloromethane andchloroform; amines such as pyridine, piperidine and triethylamine;alkylketones, such as acetone, methylethylketone and methylisobutyl;alcohols, such as methanol, ethanol and propanol; non-protonic polarsolvent, such as N,N-dimethylformamide, N,N-dimethylacetamide,acetonitrile, dimethylsulfoxide and hexamethyl phosphoric acid triamide.Among non-reactive organic solvents that are ordinarily used in theorganic synthesis, preferable solvents are those from which watergenerated in the reaction can be removed by a Dean-Stark trap. Theexamples of such solvents include, but are not limited to benzene,toluene, xylene and the like. The reaction product thus obtained may beisolated and purified by condensation, extraction and the like, which isordinarily conducted in the field of the organic synthesis, if desired,by silica gel column chromatography. The individual enantiomers of PGDHinhibitors having the formula III can be separated by a preparative HPLCusing chromatography columns containing chiral stationary phases.

Further, embodiments of this application include any modifications forthe preparation method of the 15-PGDH inhibitors described above. Inthis connection, any intermediate product obtainable from any step ofthe preparation method can be used as a starting material in the othersteps. Such starting material can be formed in situ under certainreaction conditions. Reaction reagents can also be used in the form oftheir salts or optical isomers.

Depending on the kinds of the substituents to be used in the preparationof the 15-PGDH inhibitors, and the intermediate product and thepreparation method selected, novel 15-PGDH inhibitors can be in the formof any possible isomers such as substantially pure geometrical (cis ortrans) isomers, optical isomers (enantiomers) and racemates.

In some embodiments, a 15-PGDH inhibitor having formula (III) caninclude a compound with the following formula:

and pharmaceutically acceptable salts thereof.

Advantageously, the 15-PDGH inhibitor having formula (III) was found to:i) inhibit recombinant 15-PGDH at 1 nM concentration; ii) inhibit15-PGDH in cell lines at 100 nM concentration, iii) increase PGE₂production by cell lines; iv) is chemically stable in aqueous solutionsover broad pH range; v) is chemically stable when incubated withhepatocyte extracts, vi) is chemically stable when incubated withhepatocyte cell lines; vii) shows 253 minutes plasma half-life wheninjected IP into mice; and viii) shows no immediate toxicity over 24hours when injected IP into mice at 0.6 μmole/per mouse and at 1.2μmole/per mouse and also no toxicity when injected IP into mice at 0.3μmole/per mouse twice daily for 21 days.

In other embodiments, a 15-PGDH inhibitor having formula (III) caninclude a compound with the following formula:

and pharmaceutically acceptable salts thereof.

In still other embodiments, a 15-PGDH inhibitor having formula (III) caninclude a compound with the following formula:

and pharmaceutically acceptable salts thereof.

In other embodiments, the 15-PDHG inhibitor can comprise a (+) or (−)optical isomer of a 15-PGDH inhibitor having formula (III). In stillother embodiments, the 15-PDHG inhibitor can comprise a mixture at leastone of a (+) or (−) optical isomer of a 15-PGDH inhibitor having formula(III). For example, the 15-PGDH inhibitor can comprise a mixture of:less than about 50% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (III) and greater than about 50% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (III), lessthan about 25% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (III) and greater than about 75% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (III), lessthan about 10% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (III) and greater than about 90% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (III), lessthan about 1% by weight of the (−) optical isomer of a 15-PGDH inhibitorhaving formula (III) and greater than about 99% by weight of the (+)optical isomer of a 15-PGDH inhibitor having formula (III), greater thanabout 50% by weight of the (−) optical isomer of a 15-PGDH inhibitorhaving formula (III) and less than about 50% by weight of the (+)optical isomer of a 15-PGDH inhibitor having formula (III), greater thanabout 75% by weight of the (−) optical isomer of a 15-PGDH inhibitorhaving formula (III) and less than about 25% by weight of the (+)optical isomer of a 15-PGDH inhibitor having formula (III), greater thanabout 90% by weight of the (−) optical isomer of a 15-PGDH inhibitorhaving formula (III) and less than about 10% by weight of the (+)optical isomer of a 15-PGDH inhibitor having formula (III), or greaterthan about 99% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (III) and less than about 1% by weight of the(+) optical isomer of a 15-PGDH inhibitor having formula (III).

In a still further embodiment, the 15-PDGH inhibitor can consistessentially of or consist of the (+) optical isomer of a 15-PGDHinhibitor having formula (III). In yet another embodiment, the PDGHinhibitor can consist essentially of or consist of the (−) opticalisomer of a 15-PGDH inhibitor having formula (III).

The 15-PGDH inhibitors described herein can be used for the preventionor the treatment of diseases that are associated with 15-PGDH and/ordecreased prostaglandin levels and/or where it desirable to increaseprostaglandin levels in the subject. For example, as discussed above, itis known that prostaglandins play an important role in hair growth.Specifically, internal storage of various types (A₂, F_(2a), E₂) ofprostaglandins in the various compartments of hair follicles or theiradjacent skin environments has been shown to be essential in maintainingand increasing hair density (Colombe L et. al, 2007, Exp. Dermatol,16(9), 762-9). It has been reported that 15-PGDH, which is involved inthe degradation of prostaglandins is present in the hair follicle dermalpapillae, inactivates prostaglandins, especially, PGF_(2a) and PGE₂, tocause scalp damage and alopecia (Michelet J F et. al., 2008, Exp.Dermatol, 17(10), 821-8). Thus, the compounds described herein, whichhave a suppressive or inhibitory activity against 15-PGDH that degradesprostaglandins, can improve scalp damage, prevent alopecia and promotehair growth and be used in a pharmaceutical composition for theprevention of alopecia and the promotion of hair growth.

In other embodiments, the 15-PGDH inhibitors described herein can beused in a pharmaceutical composition for promoting and/or inducingand/or stimulating pigmentation of the skin and/or skin appendages,and/or as an agent for preventing and/or limiting depigmentation and/orwhitening of the skin and/or skin appendages, in particular as an agentfor preventing and/or limiting canities.

In still other embodiments, the 15-PGDH inhibitors described herein canbe used in a pharmaceutical composition for the prevention or thetreatment of cardiovascular disease and/or diseases of vascularinsufficiency, such as Raynaud's disease, Buerger's disease, diabeticneuropathy, and pulmonary artery hypertension. Prostaglandins includingprostaglandin homologues produced in the body have been known tomaintain the proper action of the blood vessel wall, especially tocontribute to vasodilation for blood flow, preventing plateletaggregation and modulating the proliferation of smooth muscle thatsurrounds blood vessel walls (Yan. Cheng et. al., 2006, J. Clin.,Invest). In addition, the inhibition of prostaglandins production or theloss of their activity causes the degeneration of the endothelium in theblood vessel walls, platelet aggregation and the dysfunction of cellularmechanism in the smooth muscle. Among others, the production ofprostaglandins in blood vessels was shown to be decreased inhypertension patients, including pulmonary artery hypertension.

In other embodiments, the 15-PGDH inhibitors described herein can beused in a pharmaceutical composition for the prevention or the treatmentof oral and/or gastrointestinal diseases, such as oral ulcers, gumdisease, gastritis, colitis, ulcerative colitis, and gastric ulcers.Gastritis and gastric ulcer, representatives of the gastrointestinaldiseases, are defined as the conditions where gastrointestinal mucusmembrane is digested by gastric acid to form ulcer. In the stomach wallsgenerally consisting of mucosa, submucosa, muscle layer and serosa,gastric ulcer even damages submucosa and muscle layer, while gastritisdamages mucosa only. Although the morbidity rates of gastritis andgastric ulcer are relatively high, the causes thereof have not beenclarified yet. Until now, they are known to be caused by an imbalancebetween aggressive factors and defensive factors, that is, the increasein aggressive factors such as the increase in gastric acid or pepsinsecretion, or the decrease in defensive factors such as structural ormorphological deficit of the gastric mucus membrane, the decrease inmucus and bicarbonate ion secretion, the decrease in prostaglandinproduction, or the like.

Currently available therapeutic agents for gastritis and gastric ulcercomprise various drugs for strengthening the defensive factors such asan antacid, which does not affect, gastric acid secretion butneutralizes gastric acid that has been already produced, an inhibitor ofgastric acid secretion, a promoter of prostaglandin secretion, and acoating agent for stomach walls. Especially, prostaglandins are known tobe essential in maintaining the mechanism for protecting and defendinggastric mucus membrane (Wallace J L., 2008, Physiol Rev., 88(4),1547-65, S. J. Konturek et al., 2005, Journal of Physiology andPharmacology, 56(5)). In view of the above, since the 15-PGDH inhibitorsdescribed herein show a suppressive or inhibitory activity against15-PGDH, which degrades prostaglandins that protect gastric mucusmembrane, they can be effective for the prevention or the treatment ofgastrointestinal diseases, inter alia, gastritis and gastric ulcer.

In the kidney, prostaglandins modulate renal blood flow and may serve toregulate urine formation by both renovascular and tubular effects. Inclinical studies, PGE₁ has been used to improve creatinine clearance inpatients with chronic renal disease, to prevent graft rejection andcyclosporine toxicity in renal transplant patients, to reduce theurinary albumin excretion rate and N-acetyl-beta-D-glucosaminidaselevels in patients with diabetic nephropathy (see Porter, Am., 1989, J.Cardiol., 64: 22E-26E). In addition, U.S. Pat. No. 5,807,895 discloses amethod of preventing renal dysfunction by intravenous administration ofprostaglandins such as PGE₁, PGE₂ and PGI₂. Furthermore, it has beenreported that prostaglandins serve as vasodilators in the kidney, and,thus, the inhibition of prostaglandin production in the kidney resultsin renal dysfunction (Hao. C M, 2008, Annu Rev Physiol, 70,357.about.77).

Thus, the 15-PGDH inhibitors described herein, which have a suppressiveor inhibitory activity against 15-PGDH that degrades prostaglandins, maybe effective in the prevention or the treatment of renal diseases thatare associated with renal dysfunction.

The term “renal dysfunction” as used herein includes such manifestationsas follows: lower than normal creatinine clearance, lower than normalfree water clearance, higher than normal blood urea, nitrogen, potassiumand/or creatinine levels, altered activity of kidney enzymes such asgamma glutamyl synthetase, alanine phosphatidase,N-acetyl-beta-D-glucosaminidase, or beta-w-microglobulin; and increaseover normal levels of macroalbuminuria.

Prostaglandins including PGE₁, PGE₂ and PGF₂a have also been shown tostimulate bone resorption and bone formation to increase the volume andthe strength of the bone (H. Kawaguchi et. al., Clinical Orthop. Rel.Res., 313, 1995; J. Keller et al., Eur. Jr. Exp. Musculoskeletal Res.,1, 1992, 8692). Considering that 15-PGDH inhibits the activities ofprostaglandins as mentioned in the above, the inhibition of 15-PGDHactivity may lead to the promotion of bone resorption and bone formationthat are inhibited by 15-PGDH. Thus, the 15-PGDH inhibitors describedherein can be effective for the promotion of bone resorption and boneformation by inhibiting 15-PGDH activity. 15-PGDH inhibitors can also beused to increase bone density, treat osteoporosis, promote healing offractures, or promote healing after bone surgery or joint replacement.

In yet other embodiments, the 15-PGDH inhibitors described herein caneffective for treating 15-PGDH expressing cancers. Inhibition of 15-PGDHcan inhibit the growth, proliferation, and metastasis of 15-PGDHexpressing cancers.

In still other embodiments, the 15-PGDH inhibitors described herein canbe effective for wound healing. Among various prostaglandins, PGE₂ isknown to serve as a mediator for wound healing. Therefore, when skin isinjured by wounds or burns, the inhibition of 15-PGDH activity canproduce the treatment effect of the wounds or the burns by PGE₂.

Additionally, as discussed above, increased prostaglandin levels havebeen shown to stimulate signaling through the Wnt signaling pathway viaincreased beta-catenin mediated transcriptional activity. Wnt signalingis known to be a key pathway employed by tissue stem cells, andincreasing PGE₂ signaling has in model organisms been shown to increasenumbers of hematopoietic stem cells. Hence, 15-PGDH inhibitors describedherein may be utilized to increase tissue stem cell numbers for purposesthat would include increasing resistance to tissue damage by radiation,increasing resistance to environmental exposures to radiation,increasing stem cell numbers to increase fitness of bone marrow or othertypes of transplantation (through either in vivo exposure to 15-PGDHinhibitors described herein to increase stem cell numbers prior toharvest of a transplanted tissue, or through ex vivo exposure of aharvested tissue prior to transplant into a recipient host, or throughtreatment of the recipient host either before, during, or after receiptof the transplant).

In some embodiments, the 15-PGDH inhibitor can be administered to a bonemarrow graft donor or a hematopoietic stem cell donor to increase thefitness of a donor bone marrow graft or a donor hematopoietic stem cellgraft.

In other embodiments, the 15-PGDH inhibitor can also be administered tobone marrow of a subject to increase stem cells in the subject or toincrease the fitness of the marrow as a donor graft.

In still other embodiments, the 15-PGDH inhibitor can be administered toa preparation of hematopoietic stem cells, peripheral bloodhematopoietic stem cells, or umbilical cord stem cells of the subject toincrease the fitness of the stem cell preparation as a donor graft or todecrease the number of units of umbilical cord blood required fortransplantation.

In yet other embodiments, the 15-PGDH inhibitor can be administered to asubject to mitigate bone marrow graft rejection, to enhance bone marrowgraft engraftment, to enhance engraftment of a hematopoietic stem cellgraft, or an umbilical cord stem cell graft, to enhance engraftment of ahematopoietic stem cell graft, or an umbilical cord stem cell graft,and/or to decrease the number of units of umbilical cord blood requiredfor transplantation into the subject. The administration can be, forexample, following treatment of the subject or the marrow of the subjectwith radiation therapy, chemotherapy, or immunosuppressive therapy.

In other embodiments, the 15-PGDH inhibitor can be administered to arecipient of a bone marrow transplant, of a hematopoietic stem celltransplant, or of an umbilical cord stem cell transplant, in order todecrease the administration of other treatments or growth factors.

In further embodiments, the 15-PGDH inhibitor can be administered to asubject or to a tissue graft of a subject to mitigate graft rejection,to enhance graft engraftment, to enhance graft engraftment followingtreatment of the subject or the marrow of the subject with radiationtherapy, chemotherapy, or immunosuppressive therapy, to conferresistance to toxic or lethal effects of exposure to radiation, conferresistance to the toxic effect of Cytoxan, the toxic effect offludarabine, the toxic effect of chemotherapy, or the toxic effect ofimmunosuppressive therapy, to decrease infection, and/or to decreasepulmonary toxicity from radiation.

Additionally, in model organism PGE₂ signaling stimulates liverregeneration and increase survival after exposure to hepatoxic agents,such as acetaminophen. Hence, 15-PGDH inhibitors described herein may beutilized to increase liver regeneration after liver resection, or toincrease liver regeneration and increase survival after exposures tohepatoxic agents, including but not limited to acetaminophen and similarcompounds.

PGE₁ analogues have also been used in the treatment of erectiledysfunction. Accordingly, in some embodiments, 15-PGDH inhibitorsdescribed herein can used either alone or combination with aprostaglandin for the treatment of erectile dysfunction.

The 15-PGDH inhibitors described herein can be provided in apharmaceutical composition or cosmetic composition depending on thepathological or cosmetic condition or disorder being treated. Apharmaceutical composition containing the 15-PGDH inhibitors describedherein as an active ingredient may be manufactured by mixing thederivative with a pharmaceutically acceptable carrier(s) or anexcipient(s) or diluting the 15-PGDH inhibitors with a diluent(s) inaccordance with conventional methods. The pharmaceutical composition mayfurther contain fillers, anti-cohesives, lubricants, wetting agents,flavoring agents, emulsifying agents, preservatives and the like. Thepharmaceutical composition may be formulated into a suitable formulationin accordance with the methods known to those skilled in the art so thatit can provide an immediate, controlled or sustained release of the15-PGDH inhibitors after being administered into a mammal.

In some embodiments, the pharmaceutical composition may be formulatedinto a parenteral or oral dosage form. The solid dosage form for oraladministration may be manufactured by adding excipient, if necessary,together with binder, disintegrants, lubricants, coloring agents, and/orflavoring agents, to the 15-PGDH inhibitors and shaping the resultingmixture into the form of tablets, sugar-coated pills, granules, powderor capsules. The additives that can be added in the composition may beordinary ones in the art. For example, examples of the excipient includelactose, sucrose, sodium chloride, glucose, starch, calcium carbonate,kaolin, microcrystalline cellulose, silicate and the like. Exemplarybinders include water, ethanol, propanol, sweet syrup, sucrose solution,starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose,shellac, calcium phosphonate and polypyrrolidone. Examples of thedisintegrant include dry starch, sodium arginate, agar powder, sodiumbicarbonate, calcium carbonate, sodium lauryl sulfate, stearicmonoglyceride and lactose. Further, purified talc, stearates, sodiumborate, and polyethylene glycol may be used as a lubricant; and sucrose,bitter orange peel, citric acid, tartaric acid, may be used as aflavoring agent. In some embodiments, the pharmaceutical composition canbe made into aerosol formulations (e.g., they can be nebulized) to beadministered via inhalation.

The 15-PGDH inhibitors described herein may be combined with flavoringagents, buffers, stabilizing agents, and the like and incorporated intooral liquid dosage forms such as solutions, syrups or elixirs inaccordance with conventional methods. One example of the buffers may besodium citrate. Examples of the stabilizing agents include tragacanth,acacia and gelatin.

In some embodiments, the 15-PGDH inhibitors described herein may beincorporated into an injection dosage form, for example, for asubcutaneous, intramuscular or intravenous route by adding thereto pHadjusters, buffers, stabilizing agents, relaxants, topical anesthetics.Examples of the pH adjusters and the buffers include sodium citrate,sodium acetate and sodium phosphate. Examples of the stabilizing agentsinclude sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid.The topical anesthetics may be procaine HCl, lidocaine HCl and the like.The relaxants may be sodium chloride, glucose and the like.

In other embodiments, the 15-PGDH inhibitors described herein may beincorporated into suppositories in accordance with conventional methodsby adding thereto pharmaceutically acceptable carriers that are known inthe art, for example, polyethylene glycol, lanolin, cacao butter orfatty acid triglycerides, if necessary, together with surfactants suchas Tween.

The pharmaceutical composition may be formulated into various dosageforms as discussed above and then administered through various routesincluding an oral, inhalational, transdermal, subcutaneous, intravenousor intramuscular route. The dosage can be a pharmaceutically effectiveamount. The pharmaceutically effective amount can be an amount of the15-PGDH inhibitor to treat or improve alopecia, cardiovascular disease,gastrointestinal disease, wounds, and renal disease. Thepharmaceutically effective amount of the compound will be appropriatelydetermined depending on the kind and the severity of the disease to betreated, age, sex, body weight and the physical condition of thepatients to be treated, administration route, duration of therapy andthe like. Generally, the effective amount of the compound may be in therange of about 1 to 1,000 mg in the oral administration, about 0.1 to500 mg in the intravenous administration, about 5 to 1,000 mg in therectal administration. Generally, the daily dosage for adults is in therange of about 0.1 to 5,000 mg, preferably about to 1,000 mg but cannotbe determined uniformly because it depends on age, sex, body weight andthe physical condition of the patients to be treated. The formulationmay be administered once a day or several times a day with a divideddose.

Cosmetic compositions containing the 15-PGDH inhibitor can include anysubstance or preparation intended to be brought into contact with thevarious superficial parts of the human body (epidermis, body hair andhair system, nails, lips and external genital organs) or with the teethor the buccal mucous membranes for the purpose, exclusively or mainly,of cleansing them, of giving them a fragrance, of modifying theirappearance and/or of correcting body odors and/or protecting them or ofmaintaining them in good condition.

The cosmetic composition can comprise a cosmetically acceptable mediumthat may be water or a mixture of water and at least one solventselected from among hydrophilic organic solvents, lipophilic organicsolvents, amphiphilic organic solvents, and mixtures thereof.

For topical application, the cosmetic composition can be administered inthe form of aqueous, alcoholic, aqueous-alcoholic or oily solutions orsuspensions, or of a dispersion of the lotion or serum type, ofemulsions that have a liquid or semi-liquid consistency or are pasty,obtained by dispersion of a fatty phase in an aqueous phase (O/W) orvice versa (W/O) or multiple emulsions, of a free or compacted powder tobe used as it is or to be incorporated into a physiologically acceptablemedium, or else of microcapsules or microparticles, or of vesiculardispersions of ionic and/or nonionic type. It may thus be in the form ofa salve, a tincture, milks, a cream, an ointment, a powder, a patch, animpregnated pad, a solution, an emulsion or a vesicular dispersion, alotion, aqueous or anhydrous gels, a spray, a suspension, a shampoo, anaerosol or a foam. It may be anhydrous or aqueous. It may also comprisesolid preparations constituting soaps or cleansing cakes.

The cosmetic compositions may in particular comprise a hair carecomposition, and in particular a shampoo, a setting lotion, a treatinglotion, a styling cream or gel, restructuring lotions for the hair, amask, etc. The cosmetic compositions can be a cream, a hair lotion, ashampoo or a conditioner. These can be used in particular in treatmentsusing an application that may or may not be followed by rinsing, or elsein the form of a shampoo. A composition in the form of a foam, or elsein the form of spray or an aerosol, then comprising propellant underpressure, is also intended. It can thus be in the form of a lotion,serum, milk, cream, gel, salve, ointment, powder, balm, patch,impregnated pad, cake or foam.

In particular, the compositions for application to the scalp or the haircan be in the form of a hair care lotion, for example for daily ortwice-weekly application, of a shampoo or of a hair conditioner, inparticular for twice-weekly or weekly application, of a liquid or solidsoap for cleansing the scalp, for daily application, of a hairstyleshaping product (lacquer, hair setting product or styling gel), of atreatment mask, or of a foaming gel or cream for cleansing the hair.These may also be in the form of a hair dye or mascara to be appliedwith a brush or a comb.

Moreover, for topical application to the eyelashes or body hair, thecompositions may be in the form of a pigmented or unpigmented mascara,to be applied with a brush to the eyelashes or alternatively to beard ormoustache hair. For a composition administration by injection, thecomposition may be in the form of an aqueous lotion or an oilysuspension. For oral use, the composition may be in the form ofcapsules, granules, oral syrups or tablets. According to a particularembodiment, the composition is in the form of a hair cream or hairlotion, a shampoo, a hair conditioner or a mascara for the hair or forthe eyelashes.

In a known manner, the cosmetic compositions may also contain adjuvantsthat are normal in the cosmetics field, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic additives,preservatives, antioxidants, solvents, fragrances, fillers, UV-screeningagents, odor absorbers and dyestuffs. The amounts of these variousadjuvants are those conventionally used in the cosmetics field, and arefor example from 0.1% to 20%, in particular less than or equal to 10%,of the total weight of the composition. According to their nature, theseadjuvants can be introduced into the fatty phase, into the aqueous phaseand/or into the lipid spherules.

In some embodiments, the 15-PGDH inhibitor can be administered in acombinatorial therapy or combination therapy that includesadministration of a 15-PGDH inhibitor with one or more additional activeagents. The phrase “combinatorial therapy” or “combination therapy”embraces the administration of the 15-PGDH inhibitor, and one or moretherapeutic agents as part of a specific treatment regimen intended toprovide beneficial effect from the co-action of these therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined period (usually minutes, hours,days or weeks depending upon the combination selected). “Combinatorialtherapy” or “combination therapy” is intended to embrace administrationof these therapeutic agents in a sequential manner, that is, whereineach therapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample by administering to the subject an individual dose having afixed ratio of each therapeutic agent or in multiple, individual dosesfor each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissue. The therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

In some embodiments, the additional active agent can be chosen inparticular from lipoxygenase inhibitors as described in EP 648488, thebradykinin inhibitors described in particular in EP 845700,prostaglandins and their derivatives, in particular those described inWO 98/33497, WO 95/11003, JP 97-100091, JP 96-134242, the agonists orantagonists of the receptors for prostaglandins, and the nonprostanoicanalogues of prostaglandins as described in EP 1175891 and EP 1175890,WO 01/74307, WO 01/74313, WO 01/74314, WO 01/74315 or WO 01/72268.

In other embodiments, the 15-PGDH inhibitors can be administered incombination with active agents, such as vasodilators, prostanoidagonists, antiandrogens, cyclosporins and their analogues,antimicrobials, triterpenes, alone or as a mixture. The vasodilators caninclude potassium channel agonists including minoxidil and itsderivatives, aminexil and the compounds described in U.S. Pat. Nos.3,382,247, 5,756,092, 5,772,990, 5,760,043, 5,466,694, 5,438,058,4,973,474, chromakalin and diazoxide. The antiandrogens can include5.alpha.-reductase inhibitors such as finasteride and the compoundsdescribed in U.S. Pat. No. 5,516,779, cyprosterone acetate, azelaicacid, its salts and its derivatives, and the compounds described in U.S.Pat. No. 5,480,913, flutamide and the compounds described in U.S. Pat.Nos. 5,411,981, 5,565,467 and 4,910,226. The antimicrobial compounds caninclude selenium derivatives, ketoconazole, triclocarban, triclosan,zinc pyrithione, itraconazole, asiatic acid, hinokitiol, mipirocine, andthe compounds described in EP 680745, clinycine hydrochloride, benzoylor benzyl peroxide and minocycline. The anti-inflammatory agents caninclude inhibitors specific for Cox-2 such as for example NS-398 andDuP-697 (B. Batistini et al., DN&P 1994; 7(8):501-511) and/or inhibitorsof lipoxygenases, in particular 5-lipoxygenase, such as for examplezileuton (F. J. Alvarez & R. T. Slade, Pharmaceutical Res. 1992;9(11):1465-1473).

Other active compounds, which can be present in pharmaceutical and/orcosmetic compositions can include aminexil and its derivatives,60-[(9Z,12Z)octadec-9,12-dienoyl]hexapyranose, benzalkonium chloride,benzethonium chloride, phenol, oestradiol, chlorpheniramine maleate,chlorophyllin derivatives, cholesterol, cysteine, methionine, benzylnicotinate, menthol, peppermint oil, calcium panthotenate, panthenol,resorcinol, protein kinase C inhibitors, prostaglandin H synthase 1 orCOX-1 activators, or COX-2 activators, glycosidase inhibitors,glycosaminoglycanase inhibitors, pyroglutamic acid esters,hexosaccharidic or acylhexosaccharidic acids, substituted ethylenearyls,N-acylated amino acids, flavonoids, derivatives and analogues ofascomycin, histamine antagonists, triterpenes, such as ursolic acid andthe compounds described in U.S. Pat. Nos. 5,529,769, 5,468,888,5,631,282, saponins, proteoglycanase inhibitors, agonists andantagonists of oestrogens, pseudopterins, cytokines and growth factorpromoters, IL-1 or IL-6 inhibitors, IL-10 promoters, TNF inhibitors,vitamins, such as vitamin D, analogues of vitamin B12 and panthotenol,hydroxy acids, benzophenones, esterified fatty acids, and hydantoin.

Pharmaceutical and/or cosmetic compositions including the 15-PGDHinhibitor described herein can additionally contain, for example, atleast one compound chosen from prostaglandins, in particularprostaglandin PGE₁, PGE₂, their salts, their esters, their analogues andtheir derivatives, in particular those described in WO 98/33497, WO95/11003, JP 97-100091, JP 96-134242, in particular agonists of theprostaglandin receptors. It may in particular contain at least onecompound such as the agonists (in acid form or in the form of aprecursor, in particular in ester form) of the prostaglandin F₂αreceptor, such as for example latanoprost, fluprostenol, cloprostenol,bimatoprost, unoprostone, the agonists (and their precursors, inparticular the esters such as travoprost) of the prostaglandin E₂receptors such as 17-phenyl PGE₂, viprostol, butaprost, misoprostol,sulprostone, 16,16-dimethyl PGE₂, 11-deoxy PGE₁, 1-deoxy PGE₁, theagonists and their precursors, in particular esters, of theprostacycline (IP) receptor such as cicaprost, iloprost,isocarbacycline, beraprost, eprostenol, treprostinil, the agonists andtheir precursors, in particular the esters, of the prostaglandin D₂receptor such as BW245C((4S)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-anoicacid), BW246C((4R)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-anoicacid), the agonists and their precursors, in particular the esters, ofthe receptor for the thromboxanes A2 (TP) such as I-BOP ([1S-[1a,2a(Z),3b(1E,3S),4a]]-7-[3-[3-hydroxy-4-[4-(iodophenoxy)-1-butenyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoicacid).

Advantageously, the composition can include at least one 15-PGDHinhibitor as defined above and at least one prostaglandin or oneprostaglandin derivative such as for example the prostaglandins ofseries 2 including in particular PGF₂a and PGE₂ in saline form or in theform of precursors, in particular of the esters (example isopropylesters), their derivatives such as 16,16-dimethyl PGE₂, 17-phenyl PGE₂and 16,16-dimethyl PGF_(2α) 17-phenyl PGF_(2α), prostaglandins of series1 such as 11-deoxyprostaglandin E1, 1-deoxyprostaglandin E1 in saline orester form, is their analogues, in particular latanoprost, travoprost,fluprostenol, unoprostone, bimatoprost, cloprostenol, viprostol,butaprost, misoprostol, their salts or their esters.

In accordance with another aspect of the application, the modulator of15-PGDH can be a 15-PGDH activator that can promote or stimulate theactivity of 15-PGDH. In certain embodiments, the 15-PDGH activator caninclude a compound having the formula (IV):

-   -   wherein X₃ and Y₂ are independently C or SO;    -   U is OR″(wherein R″ is H, a substituted or unsubstituted alkyl        group, or substituted or unsubstituted aryl group) or

-   -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each selected from the group        consisting of H, F, Cl, Br, I, an alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′        (wherein R′ is H or a lower alkyl group), a substituted or        unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a        substituted or unsubstituted heterocyclyl, and R₈ and R₉ may be        linked to form a cyclic or polycyclic ring; and pharmaceutically        acceptable salts thereof.

In other embodiments, the 15-PDGH activator can include a compoundhaving the formula (V):

-   -   wherein U is OR″(wherein R″ is H, a substituted or unsubstituted        alkyl group, or substituted or unsubstituted aryl group) or

-   -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each selected from the group        consisting of H, F, Cl, Br, I, an alkyl group, (CH₂)_(n1)OR′        (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,        CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or I), CN,        (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′        (wherein R′ is H or a lower alkyl group), a substituted or        unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a        substituted or unsubstituted heterocyclyl, and R₈ and R₉ may be        linked to form a cyclic or polycyclic ring; and pharmaceutically        acceptable salts thereof.

In certain embodiments, a 15-PGDH activator having formula (IV) or (V)can be selected that can: ia) at 7.5 μM concentration, stimulate aVaco503 reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 50 (using a scaleon which a value of 100 indicates a doubling of reporter output overbaseline); iia) at 7.5 μM concentration stimulate a V9m reporter cellline expressing a 15-PGDH luciferase fusion construct to a luciferaseoutput level of greater than 50; iiia) at 7.5 μM concentration stimulatea LS174T reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 50; iva) at 7.5μM concentration, does not activate the negative control V9m cell lineexpressing TK-renilla luciferase reporter to a level any greater than25; and va) against recombinant 15-PGDH protein the compound shows anIC50 concentration for inhibiting 15-PGDH enzyme activity of greaterthan 2.5 μM.

In certain embodiments, a 15-PGDH activator having formula (IV) or (V)can be selected that can: ib) at 7.5 μM concentration, stimulate aVaco503 reporter cell line expressing a 15-PGDH luciferase fusionconstruct to increase luciferase output; iib) at 7.5 μM concentrationstimulate a V9m reporter cell line expressing a 15-PGDH luciferasefusion construct to increase luciferase output; iiib) at 7.5 μMconcentration stimulate a LS174T reporter cell line expressing a 15-PGDHluciferase fusion construct to increase luciferase output; ivb) at 7.5μM concentration, does not activate the negative control V9m cell lineexpressing TK-renilla luciferase reporter to a luciferase level anygreater than 25% above; and vb) against recombinant 15-PGDH protein thecompound shows an IC50 concentration for inhibiting 15-PGDH enzymeactivity of greater than or equal to 2.5 μM.

In other embodiments, a 15-PGDH activator having formula (IV) or (V)that meets the above noted criteria (ia-va) and/or that meet the abovenoted criteria (ib-vb) includes a compound having the formula (VI):

and pharmaceutically acceptable salts thereof.

In other embodiments, the 15-PGDH activator can be an analogue of acompound having the formula (VI). Such analogues can have the followingformula (VII):

-   -   wherein U is OR″(wherein R″ is H, a substituted or unsubstituted        alkyl group, or substituted or unsubstituted aryl group) or

-   -   R₈ and R₉ are each selected from the group consisting of H, F,        Cl, Br, I, an alkyl group, (CH₂)_(n1)OR′ (wherein n1=1, 2, or        3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein        X═F, Cl, Br, or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,        O(CO)R′, OR′, SR′, COOR′ (wherein R′ is H or a lower alkyl        group), a substituted or unsubstituted aryl, a substituted or        unsubstituted cycloalkyl, a substituted or unsubstituted        heterocyclyl, and R₈ and R₉ may be linked to form a cyclic or        polycyclic ring; and pharmaceutically acceptable salts thereof.

Examples of 15-PGDH activators having the formula (VII) include:

and pharmaceutically acceptable salts thereof.

Other examples of compounds having formula (VII) include:

and pharmaceutically acceptable salts thereof.

In other embodiments, the 15-PGDH activator can be an analogue ofcompound (VI) having the following formula (VIII):

-   -   wherein R₁₀ is selected from the group consisting of a        substituted or unsubstituted aryl, a substituted or        unsubstituted cycloalkyl, and a substituted or unsubstituted        heterocyclyl; and pharmaceutically acceptable salts thereof.

Examples of 15-PGDH activators having the formula (VIII) include:

and pharmaceutically acceptable salts thereof.

Still other examples of compounds having the formula (VIII) include:

and pharmaceutically acceptable salts thereof.

In still other embodiments, the 15-PGDH activator can be an analogue ofcompound (VI) having the formula (IX):

-   -   wherein R₁₁ is H, F, Cl, Br, I, a lower alkyl group,        (CH₂)_(n1)OR′ (wherein n1=1, 2, or 3), CF₃, CH₂—CH₂X,        O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂X (wherein X═F, Cl, Br, or        I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′,        COOR′ (wherein R′ is H or a lower alkyl group), a substituted or        unsubstituted aryl, a substituted or unsubstituted cycloalkyl,        and a substituted or unsubstituted heterocyclyl; and        pharmaceutically acceptable salts thereof.

Examples of 15-PGDH activators having the formula (IX) include:

and pharmaceutically acceptable salts thereof.

Still other examples of compounds having formula IX) include:

and pharmaceutically acceptable salts thereof.

In other embodiments, the 15-PGDH activator can be an analogue ofcompound (IV) having the following formulas:

and pharmaceutically acceptable salts thereof.

The 15-PGDH activators described herein can be used for the preventionor the treatment of diseases that are associated with decreased 15-PGDHlevels and/or increased prostaglandin levels. Increasing tissue levelsof 15-PGDH should decrease tissue levels of prostaglandins. Activitiesassociated with compounds that decrease tissue prostaglandins includedecreasing development of human tumors. For example, administration of15-PGDH activators can be used to treat patients with colon neoplasia,e.g., colon cancer or colon adenoma, or to treat and prevent new diseasein patients with a history of colon neoplasia, or to reverse resistanceto NSAID therapy for neoplasia therapy or neoplasia preventive therapy.Further, administration of 15-PGDH activators described herein can beused to treat subjects having an NSAID-responsive condition. In certainembodiments, 15-PGDH activators enhance NSAID-responsiveness in subjectswho are relatively unresponsive to NSAID treatment.

The 15-PGDH activators described herein can be also be used in a methodof treating any NSAID-responsive condition. The NSAID-responsivecondition applies to a subject who is NSAID-resistant or a subject whowas determined to be resistant to NSAID therapy. In the method, atherapeutically effective amount of 15-PGDH activators can beadministered alone or in combination with an effective an effectiveamount of 15-PGDH protein, cDNA, or an active fragment thereof. Thepatient may be a subject at risk of developing colon neoplasia (e.g.,based on family history), or a subject at risk of colon adenoma relapse,but is suspected of being resistant to NSAID therapy. Further, thepatient may be any subject who is undergoing or about to undergo NSAIDtherapy for any NSAID-responsive condition, but who experiences NSAIDresistance.

The 15-PGDH activators described herein can be provided in apharmaceutical composition that includes pharmaceutically acceptablecarrier. In some embodiments, the 15-PGDH activator can be providedalone or in combination with other components (e.g., an NSAID), can bemade into aerosol formulations (i.e., they can be “nebulized”) to beadministered via inhalation. The 15-PGDH activator can also be providedalone or in combination with other components in aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Compositions including the 15-PGDH activator can beadministered, for example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations can be presentedin unit-dose or multi-dose sealed containers, such as ampoules andvials. Solutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described. The modulatorscan also be administered as part of a prepared food or drug.

The dose administered to a patient should be sufficient to induce abeneficial response in the subject over time. The optimal dose level forany patient will depend on a variety of factors including the efficacyof the specific modulator employed, the age, body weight, physicalactivity, and diet of the patient, on a possible combination with otherdrugs, and on the severity of the case of diabetes. It is recommendedthat the daily dosage of the 15-PGDH activator can be determined foreach individual patient by those skilled in the art. The size of thedose also will be determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularcompound in a particular subject.

In some embodiments, the 15-PGDH activator can be administered in acombination therapy includes administration of a single pharmaceuticaldosage formulation that contains a 15-PGDH activator and one or moreadditional active agents, as well as administration of a 15-PGDHactivator and each active agent in its own separate pharmaceuticaldosage formulation. For example, a 15-PGDH activator and celecoxib canbe administered to the human subject together in a single oral dosagecomposition, such as a tablet or capsule, or each agent can beadministered in separate oral dosage formulations. In other embodiments,an NSAID, e.g., celecoxib or aspirin, may be administered with aneffective amount of the 15-PGDH activator. Where separate dosageformulations are used, a 15-PGDH activator and one or more additionalactive agents can be administered at essentially the same time (i.e.,concurrently), or at separately staggered times (i.e., sequentially).Combination therapy is understood to include all these regimens.

The invention is further illustrated by the following example, which isnot intended to limit the scope of the claims.

Example 1

This Example describes the activities of four compounds with respect tothe enzyme 15-Prostaglandin Dehydrogenase (15-PGDH) (encoded by the geneHPGD). The compounds are SW033291, SW054384, SW124531, SW145753 and havethe following formulas:

FIG. 1 shows that SW033291, SW054384, and SW145753 all increaseluciferase activity of cells the express a 15-PGDH luciferase fusionconstruct created by targeted gene knock-in of renilla luciferase intothe last coding exon of 15-PGDH. The activity is demonstrated in threedifferent colon cancer cell lines all engineered to contain the15-PGDH-luciferase fusion. These cell lines are Vaco-9m (V9m), LS174T,Vaco503 (V503). SW054384 is in general the best inducer, and showsmaximum activity at 6.25 μM. Value of 1.0 on the Y-axis is the basallevel of reporter activity in cells treated with drug free DMSO vehicle.

FIG. 2 shows western blots demonstrating that SW033291, SW054384, andSW145753 all increase levels of 15-PGDH protein in cell lines V503,LS174T, and V503 treated with 7.5 μM compound for 48 hours. UntreatedFET cells provide a positive control for 15-PGDH expression.

FIG. 3 shows western blot demonstrating SW124531 also increases 15-PGDHprotein levels in colon cell lines (FET cells treated with TGF-ß (10ng/ml) for 48 hours are used as a positive control for 15-PGDHexpression in certain panels).

FIG. 4 shows western blot demonstrating 5 μM SW124531 increases levelsof 15-PGDH protein (wt-PGDH) expressed from a cDNA expression vector inV400-S3-2-32 cells, and also increases protein levels of a catalyticallydead mutant 15-PGDH (mu-PGDH) also expressed from a cDNA expressionvector in V400-M3-2-72 cells. As these proteins are expressed from aheterologous CMV promoter, the findings suggest that the compounds workdirectly on stabilizing the 15-PGDH protein. The compounds show noeffects on levels of a related enzyme, 17-beta-estradiol-dehydrogenase.

FIG. 5 shows increase in 15-PGDH protein levels in V503 cells treatedwith SW124531 as assayed by immuno-fluorescence (upper two rows) and bywestern blot (lower panel).

FIGS. 6-9 show that SW033291, SW054384, SW145753, and SW124531 do not ingeneral alter 15-PGDH mRNA levels in treated colon cancer cell lines asassessed by real-time PCR. The only exception is the slight increase in15-PGDH mRNA in SW033291 treated V503 cells, which is less than theinduction of 15-PGDH protein as well as 15-PGDH-luciferase reporterlevels seen in SW033291 treated V503 cells. In these studies parentalcell lines (not containing the 15-PGDH-luciferase reporter) areemployed.

FIG. 10 shows the effects of three compounds on total 15-PGDH activityin cell lines treated with the compounds. Cell lines were treated withcompounds at 7.5 μM for 48 hours, and then pelleted. Pellets were lysedand total 15-PGDH activity measured and normalized to 1,000,000 inputcells per pellet. 15-PGDH activity was assayed by measuring the transferof tritium from 15(S)-[15-3H] PGE₂ to glutamate by coupling 15-PGDH withglutamate dehydrogenase as described in (Chi X, Freeman B M, Tong M,Zhao Y, Tai H H. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) isup-regulated by flurbiprofen and other non-steroidal anti-inflammatorydrugs in human colon cancer HT29 cells. Arch Biochem Biophys. 2009;487(2):139-45.). Activity is measured as pmol PGE₂/min/million cells. Asshown, SW033291 markedly inhibits 15-PGDH activity in all three of thecell lines tested. We conclude that although SW033291 increases total15-PGDH protein levels in cells, it also inactivates 15-PGDH enzymeactivity.

In contrast, 15-PGDH enzyme activity is increased in cells treated withSW054384 and in cells treated with SW145753.

FIG. 11 shows the effect on activity of recombinant 15-PGDH protein (a15-PGDH-GST fusion protein) incubated with varying concentrations of thetest compounds, with 15-PGDH activity across a range of compoundconcentrations recorded on the table and displayed on the correspondinggraphs. As shown, SW033291 is a potent inhibitor of 15-PGDH activity,with an IC50 of <1.25 nM. This contrasts with the IC50 of between 25nM-62.5 nM measured for the commercial 15-PGDH inhibitor available fromCayman Chemical (Cayman catalogue item 10638, Cayman Chemical number13695).

FIG. 11 also shows that at very high concentration SW054384 can inhibitrecombinant 15-PGDH activity, with an IC50 of between 5 μM-50 μM. Weconclude that SW054384 increases total 15-PGDH level and activity incells treated with 7.5 μM compound, but can inhibit in vitro recombinant15-PGDH protein in vitro assays using 5 μM-50 μM compound.

FIG. 11 also shows that SW145753 can inhibit activity of recombinant15-PGDH enzyme in an in vitro assay at an IC50 between 12-6.25 nM. Thissuggests the activity of SW145753 in increasing versus in inhibiting15-PGDH activity may be discordant in cells versus in the in vitro assay(perhaps due to washout of drugs when washing the cells), or may beconcentration dependent.

FIG. 12 shows repeat testing of the effects of SW033291 and SW054384 onactivity of recombinant 15-PGDH protein tested in vitro. Assays weredone by measuring the transfer of tritium from 15(S)-[15-3H] PGE₂ toglutamate (at 1 μM PGE₂ substrate) shown at left (panels A, C), or bydirect fluorescence monitoring of NADH generation by 15-PGDH (done at 20μM PGE₂ substrate) shown at right (panels B, D). SW033291 is againconfirmed as a highly potent 15-PGDH inhibitor with an IC50 of 0.7 nM asmeasured in the tritium assay and an IC50 of 1.6 nM as measured in thefluorescence assay. The relative insensitivity of the IC50 to substrateconcentration suggests that SW033291 is a non-competitive inhibitor of15-PGDH.

SW054384 shows very weak inhibitory activity, with IC50s that are 10,000fold higher than that of SW033291 (8.4 μM and 11 μM in the tritium andfluorescent based assays respectively). This is consistent with theactivity of SW054384 being on balance to increase 15-PGDH protein leveland enzyme activity in cells.

FIG. 13 shows results of assays of 15-PGDH activity using the tritiummethod in cells treated with SW124531 (upper panel) and in recombinant15-PGDH protein treated with SW124531 (lower panel). SW124531 showsactivity in increasing 15-PGDH activity in most cell lines, though thisactivity is best in cell lines in which basal 15-PGDH activity is >10units. SW124531 also inhibits activity of 15-PGDH recombinant protein atan IC50 of 50 nM.

FIG. 14 shows assay of different compounds for ability to directly bindto recombinant 15-PGDH protein as measured by shifting the meltingtemperature of the protein. The melting of the protein is followed bymeasurement of the fluorescence of SYPRO Orange dye (Sigma #S5692) thatincreases as the dye binds to hydrophobic residues exposed as theprotein melt. The graph at upper left shows the melt curves of 15-PGDHwith all of the assays done in the presence of the different compoundssuperimposed on each other. The graph at upper right plots the negativederivative of fluorescence versus temperature for each of the curvesshown at left, with the melting point measured as the temperature of thenegative peak (i.e., the point of most rapid change in the fluorescenceversus temperature plot). The results are shown in tabular form on thetable below. Lapatinib is used as a negative control. There is nobinding of any drug in the absence of enzyme co-factor (either NAD orNADH). In the presence of either NAD or NADH, SW033291 creates two peaksin the melting curve, with one of these peaks displaced by 15 degreesCelsius, consistent with SW033291 binding directly to 15-PGDH. SW124531and SW145753 also show evidence of direct binding to 15-PGDH. In thisassay, SW054384 cannot be demonstrated to bind 15-PGDH. It is possiblethat SW054384 does bind to 15-PGDH, but that the binding is weak and ismelted off at a temperature below the melting temperature of the 15-PGDHprotein. Assays were done at both 10 μM and 100 μM cofactor (testingboth NAD and NADH), which compares well with the published Km of NAD of15.8 μM.

FIG. 15 shows that none of the four compounds tested induce a shift inthe melting temperature of catalytically inactive mutant 15-PGDHprotein. We interpret the induction of 15-PGDH mutant protein bySW124531 as suggesting that SW124531 likely has weak binding to mutant15-PGDH that is able to stabilize protein at 37° C., but with the drugmelted off at a temperature below 50° C., that is the meltingtemperature of the protein.

FIG. 16 shows the in vivo modulation by compounds of 15-PGDH activity asreflected in PGE₂ levels that are assayed in the medium of A549 cellsthat have been stimulated by IL1-beta for 23 hours, with compound addedfor the last 5 hours (blue bars). The increment in PGE₂ level shows theclear inhibition of 15-PGDH activity in the cells by addition ofSW033291 (as well as SW145753, SW124531, and a commercial 15-PGDHinhibitor from Cayman Chemical. In an additional iteration (redbars)(2), SW054384 was added commencing 24 hours before addition ofIL1-beta, and then maintained for the next 26 hours in the presence ofIL1-beta. The lower level of PGE₂ produced supports that in these cellsSW054384 increased the 15-PGDH activity. Panel at left shows raw data;whereas, panel at right shows data normalized for cell numbers presentat end of the experiment. PGE₂ levels are assayed by ELISA.

FIG. 17 shows the dose response of effect on SW033291 on PGE₂ productionfrom IL1-beta treated A549 cells, as reflected in PGE₂ levels that areassayed in the medium of A549 cells that have been stimulated byIL1-beta for 24 hours, with SW033291 added for the last 8 hours.

FIG. 18 shows the in vivo modulations by 2.5 μM compounds of 15-PGDHactivity as reflected in PGE₂ levels following addition of PGE₂ into themedium of Vaco-503 cells. In this study cells are treated with compoundfor 24 hours after which PGE₂ is added into the medium. After an added24 hours PGE₂ levels remaining in the medium are assayed by Elisa. Datalabeled “medium” is a control lane with PGE₂ added to medium alone, inthe absence of cells. Data labeled DMSO is a control in which cells aretreated with DMSO only (the diluent for the compounds). The differencebetween the “medium” and the “DMSO” lanes represents the cell dependentdegradation of PGE₂ by 15-PGDH. Again demonstrated, is the near completeblockade of 15-PGDH activity by addition of 2.5 μM SW033291, asreflected by the blockade in PGE₂ degradation. Additionally demonstratedis the stimulation of 15-PGDH activity by SW054384, as reflected by theincreased degradation of PGE₂.

FIG. 19 shows the activity of 2.5 μM SW033291 in speeding the healing ofa model wound consisting of a scratch in a monolayer of HaCaT cellsobserved over 48 hours of treatment. TGF-beta serves as the positivecontrol in the assay.

FIG. 20 shows the quantitation of the width of the scratch at 0 and 48hours in the control, 2.5 μM SW033291 treated cells, and the TGF-beta (1ng/ml) treated cells.

Example 2 Analysis of Analogues of Lead Compounds SW033291, a 15-PGDHInhibitor

This Example provides data on a group of structural analogues ofSW033291. Data provided includes level of induction of a15-PGDH-luciferase fusion gene reporter, recorded as % increasedluciferase activity over basal level, in three colon cancer cell lines,V9m, V503, and LS174T, engineered to contain the reporter, and treatedwith either 2.5 μM or 7.5 μM compound (i.e., Values are recorded on ascale where 100 indicates of doubling of luciferase activity overbaseline level). Also recorded is the IC50 of each compound forinhibiting enzymatic activity of recombinant 15-PGDH in an in vitroassay.

TABLE 2 V9M V9M LS174T LS174T V503 V503 Enzyme reporter reporterreporter reporter reporter reporter inhibition activity activityactivity activity activity activity (IC₅₀, (2.5 (7.5 (2.5 (7.5 (2.5 (7.5Structures ID nM) uM) uM) uM) uM) uM) uM)

SW033291 1.23 nM 98.24 93.16 123.46 106.73 126.32 99.34

SW033291 isomer B 0.76 nM Note: Structures shown are for illustrativepurposes. We don′t know which structure corresponds to Isomer A or B.Residual activity of isomer A may be due to small amounts of Isomer Bpresent in the preparation.

SW033291 isomer A 56.56 nM Note: Structures shown are for illustrativepurposes. We don′t know which structure corresponds to Isomer A or B.Residual activity of isomer A may be due to small amounts of Isomer Bpresent in the preparation.

SW033292 1.51

413423 −5.65 −8.76 3.41 3.91 7.89 3.13

980653 8.83 11.96 5.76 10.99 −10.47 −15.21

405320 8.77 −15.45 9.95 −2.57 −7.73 −33.68

SW208078 25 nM 36.16 34.90 72.71 40.01 87.73 83.77

SW208079 125 nM 36.01 32.75 53.42 43.83 85.29 61.82

SW033290 525 nM −34.33 −25.45 −0.24 −6.58 30.93 17.71

SW208080 2.64 nM 102.08 98.65 117.81 116.64 103.70 127.19

SW208081 18 nM 37.79 63.56 64.53 95.14 90.46 105.37

SW206976 >7.5 uM 11.80 17.51 42.38 20.56 16.79 49.53

SW206977 >7.5 uM 3.5028 0.45 34.88 29.35 33.00 37.62

SW206978 >7.5 uM 7.5141 8.02 31.44 26.19 38.84 35.75

SW206979 >7.5 uM −12.59 −20.62 34.26 32.79 21.66 42.92

SW206980 0.97 nM 99.37 92.71 117.24 92.15 129.57 108.51

SW206992 1.411 nM 86.44 121.75 85.81 72.03 161.20 145.39

SW208064 151.4 nM 82.58 50.11 126.18 96.23 126.18 96.23

SW208065 4.865 nM 120.19 118.92 73.50 87.74 73.50 87.74

SW208066 1.368 nM 122.72 111.63 123.89 93.74 123.89 93.74

SW208067 2.395 nM 121.69 108.47 94.30 79.63 94.30 79.63

SW208068 >7.5 uM 12.90 12.35 14.90 15.00 14.90 15.00

SW208069 >7.5 uM −14.48 0.23 19.47 15.13 19.47 15.13

SW208070 >7.5 uM 22.56 12.11 19.69 15.43 19.69 15.43

We first note that the 15-PGDH inhibitory activity of SW033291 is atleast 98% due to the activity of one of the two optical isomers of thiscompound, designated isomer A and B. The structural assignment of isomerA versus isomer B is not yet established.

There is an important effect on the length of the carbon side chain ofSW033291 on the IC50 for inhibiting recombinant 15-PGDH in vitro.Compared to SW033291 (4 carbons): SW208080 (5 carbons) has IC50 1.5times higher, SW208081 (6 carbons) has IC50 10 times higher, andSW208079 (1 carbon) has IC50 over 60-fold higher, with marked loss ofactivity in inducing the cell line reporters.

The sulfoxide group appears to be a critical substituent, as inactivesubstitutions of the sulfoxide include the corresponding: ketone(SW206976), amide (SW206977), ester (SW206978), and carboxylic acid(SW206979). However, inhibitory activity is observed for the sulfoneanalogs.

Deletion of the phenyl group on SW033291 (SW206980) lowers the IC50 byhalf. SW206980 continues to be a highly active compound in reporterinduction when applied to reporter cell lines at 2.5 μM concentration.

Example 3

The following Example describes the synthesis of SW033291 and analoguesthereof as well as provides mass spectrometry NMR confirmation of thestructures.

SW0332912-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using procedure describe by Kalugin. To the solution of4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile(0.53 mmol, 220 mg) in DMF (0.25 M)/EtOH (0.5 M) was added KOH (0.32mmol, 18 mg, 0.6 equiv., 0.1 M in water). The reaction mixture wasstirred at 35° C. for 40 min. Once complete, the reaction was dilutedwith EtOAc and washed with 10% aq. solution of acidic acid, the organicphase was separated and aqueous layer was extracted twice with EtOAc,dried over magnesium sulfate, filtered and concentrated under reducedpressure to give 211 mg of SW0332912-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine(96%). ¹H NMR (400 MHz, CDCl₃) δ 7.67-7.60 (m, 1H), 7.57-7.35 (m, 7H),7.10 (dd, J=5.0, 3.7 Hz, 1H), 4.54 (s, 2H), 3.26 (ddd, J=12.8, 9.1, 6.0Hz, 1H), 3.09 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 1.83-1.61 (m, 2H),1.53-1.38 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). EST-MS (m/z): 413 [M+H]⁺.

2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Acetic Acid (900 μL) and hydrogen peroxide (0.57 mmol, 1.5 equiv., 30%solution in water) were added to the solution of2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(0.38 mmol, 150 mg) in chloroform (900 μL). The reaction mixture wasstirring at 32° C. for 45 min. The reaction was then diluted with EtOAcand washed with saturated NaHCO₃ solution, dried over magnesium sulfate,filtered and concentrated under reduced pressure to give 153 mg ofdesigned product (98%). ¹H NMR (400 MHz, CDCl₃) δ 7.75 (dd, J=3.8, 1.1Hz, 1H), 7.66-7.57 (m, 2H), 7.58-7.51 (m, 4H), 7.47 (s, 1H), 7.16 (dd,J=5.0, 3.8 Hz, 1H), 4.74 (d, J=13.0 Hz, 1H), 4.41 (d, J=13.0 Hz, 1H),2.97 (dt, J=13.0, 8.2 Hz, 1H), 2.81 (dt, J=12.9, 7.3 Hz, 1H), 1.94-1.76(m, 2H), 1.53-1.38 (m, 2H), 0.94 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 413[M+H]⁺

2-(((butylthio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile. Amixture of4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.34 mmol, 101 mg), butyl(chloromethyl)sulfane (0.34 mmol, 48 mg, 1.0equiv.) and Et₃N (0.51 mmol, 72 μL, 1.5 equiv.) was refluxed in dryCH₃CN (350 μL) for 20 min. The reaction mixture was then diluted withEtOAc and water. The organic phase was separated and aqueous layer wasextracted twice with EtOAc. The combined extractions were washed withsaturated NaCl solution, dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography to give 124 mg of designed product (92%). ¹H NMR (400MHz, CDCl₃) δ 7.70 (dd, J=3.8, 1.1 Hz, 1H), 7.64-7.56 (m, 1H), 7.55-7.47(m, 5H), 7.40 (d, J=1.1 Hz, 1H), 7.14 (dd, J=5.0, 3.8 Hz, 1H), 4.53 (s,2H), 2.74 (t, J=8.0 Hz, 2H), 1.72-1.57 (m, 2H), 1.49-1.34 (m, 2H), 0.90(t, J=7.4 Hz, 3H). ESI-MS (m/z): 397 [M+H]⁺.

4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile.To a solution of 3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one (2.34 mmol,500 mg) and cyanothioacetamide (7.0 mmol, 717 mg, 3.0 equiv.) in ethanol(7 mL), a few drops of piperidine were added. The reaction was refluxedfor 3 h. The solid that formed was collected and recrystallized fromacetic acid to give designed product in 46% isolated yield. ¹H NMR (400MHz, DMSO-d₆) δ 8.17 (d, J=3.8 Hz, 1H), 7.96 (d, J=5.0 Hz, 1H),7.74-7.62 (m, 2H), 7.54 (dd, J=5.1, 2.0 Hz, 3H), 7.31-7.19 (m, 1H), 7.01(s, 1H). ESI-MS (m/z): 295 [M+H]⁺.

3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one was prepared from benzaldehydeand 1-(thiophen-2-yl)ethanone via aldol condensation using proceduredescribed by Azam showed in Scheme 2. ¹H NMR (400 MHz, CDCl₃) δ7.88-7.80 (m, 2H), 7.67 (dd, J=4.9, 1.1 Hz, 1H), 7.66-7.59 (m, 2H),7.47-7.34 (m, 4H), 7.18 (dd, J=5.0, 3.8 Hz, 1H). ESI-MS (m/z): 215[M+H]⁺.

3-phenyl-1-(thiazol-2-yl)prop-2-en-1-one was prepared from via Wittigreaction using procedure described by Merino showed in Scheme 2. ¹H NMR(400 MHz, Chloroform-d) δ 8.06 (d, J=3.0 Hz, 1H), 7.99 (s, 1H), 7.96 (s,1H), 7.75-7.67 (m, 3H), 7.44-7.38 (m, 3H). ESI-MS (m/z): 216 [M+H]⁺.

SW208079-1-A2-(methylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW033291 and showed in Scheme 1 and 2. ¹H NMR (500 MHz, CDCl₃)δ 7.67-7.50 (m, 5H), 7.50-7.36 (m, 3H), 7.16-7.09 (m, 1H), 4.58 (s, 2H),2.99 (s, 3H). ESI-MS (m/z): 371 [M+H]⁺.

SW208080-1-A2-(pentylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW033291 and showed in Scheme 1 and 2. ¹H NMR (500 MHz,CD₂Cl₂) δ 7.98-7.36 (m, 8H), 7.33-6.85 (m, 1H), 4.47 (s, 2H), 3.28-3.15(m, 1H), 3.09-2.99 (m, 1H), 1.81-1.59 (m, 2H), 1.50-1.25 (m, 4H), 0.88(t, J=7.2 Hz, 3H). ESI-MS (m/z): 427 [M+H]⁺.

SW208081-1-A2-(hexylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW033291 and showed in Scheme 1 and 2. ¹H NMR (500 MHz,CD₂Cl₂) δ 7.78-7.66 (m, 1H), 7.63-7.46 (m, 7H), 7.27-7.02 (m, 1H), 4.11(s, 2H), 3.43-3.20 (m, 1H), 3.11 (ddd, J=13.8, 9.4, 6.4 Hz, 1H),1.89-1.63 (m, 2H), 1.58-1.39 (m, 4H), 1.40-1.21 (m, 2H), 0.91 (d, J=6.8Hz, 3H). ESI-MS (m/z): 441 [M+H]⁺.

SW208066,2-(butylsulfinyl)-4-phenyl-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW033291 and showed in Scheme 1 and 2. ¹H NMR (400 MHz, CDCl₃)δ 8.06 (s, 1H), 7.92 (d, J=3.2 Hz, 1H), 7.65-7.39 (m, 6H), 4.63 (s, 2H),3.28 (ddd, J=12.8, 9.0, 6.2 Hz, 1H), 3.11 (ddd, J=12.8, 9.0, 6.8 Hz,1H), 1.85-1.63 (m, 2H), 1.56-1.42 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 414 [M+H]⁺.

SW206980,2-(butylsulfinyl)-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine wasprepared by using synthetic procedures described for the preparation ofanalog SW033291 and showed in Scheme 1 and 2. ¹H NMR (400 MHz, CDCl₃) δ7.79 (d, J=8.5, 1H), 7.65-7.49 (m, 2H), 7.39 (dt, J=5.1, 0.7 Hz, 1H),7.06 (dd, J=5.0, 3.7, Hz, 1H), 5.20 (s, 2H), 3.26 (ddd, J=12.8, 9.0, 6.2Hz, 1H), 3.10 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 1.78-1.60 (m, 2H),1.55-1.39 (m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 337 [M+H]⁺

SW206992, 2-(butylsulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW033291 and showed in Scheme 1 and 2. ¹H NMR (400 MHz, CDCl₃)δ 8.15 (d, J=8.5 Hz, 1H), 7.95 (d, J=8.5 Hz, 1H), 7.90 (d, J=3.2 Hz,1H), 7.44 (d, J=3.2 Hz, 1H), 3.29 (ddd, J=12.7, 9.0, 6.2 Hz, 1H), 3.13(ddd, J=12.8, 9.0, 6.7 Hz, 1H), 1.83-1.61 (m, 2H), 1.59-1.38 (m, 2H),0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 338 [M+H]⁺.

SW208064, 2-(butylsulfinyl)thieno[2,3-b]pyridin-3-amine was prepared byusing synthetic procedures described for the preparation of analogSW033291 and showed in Scheme 1. ¹H NMR (400 MHz, CDCl₃) δ 8.61 (dd,J=4.7, 1.6 Hz, 1H), 7.89 (dd, J=8.1, 1.6 Hz, 1H), 7.33 (dd, J=8.1, 4.6Hz, 1H), 3.39-3.18 (m, 1H), 3.20-3.03 (m, 1H), 1.74 (p, J=7.6 Hz, 2H),1.63-1.38 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 255 [M+H]⁺.

SW208078-1-A2-(butylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Acetic Acid (50 μL) and hydrogen peroxide (0.036 mmol, 1.5 equiv., 30%solution in water) were added to the solution of SW0332912-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine(0.024 mmol, 10 mg) in chloroform (50 μL). The reaction mixture wasstirring at 32° C. for 4 h. The reaction was diluted with EtOAc andwashed with saturated NaHCO₃ solution, dried over magnesium sulfate,filtered and concentrated under reduced pressure to give crude2-(butylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine,which was purified by flash chromatography in 8% isolated yield. ¹H NMR(400 MHz, CDCl₃) δ 7.71 (d, J=3.8 Hz, 1H), 7.64-7.54 (m, 3H), 7.53-7.42(m, 4H), 7.15 (dd, J=5.0, 3.7 Hz, 1H), 5.09 (s, 2H), 3.38-3.02 (m, 2H),1.92-1.67 (m, 2H), 1.52-1.28 (m, 2H), 0.92 (t, J=7.4 Hz, 3H). ESI-MS(m/z): 429 [M+H]⁺.

SW033290-2-A2-(methylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared by using synthetic procedures described for the preparationof analog SW208078-1-A and showed in Scheme 1 and 2. ¹H NMR (500 MHz,CDCl₃) δ 7.78-7.68 (m, 1H), 7.64-7.54 (m, 3H), 7.53-7.45 (m, 4H),7.18-7.10 (m, 1H), 5.08 (s, 2H), 3.14 (s, 3H). ESI-MS (m/z): 387 [M+H]⁺.

SW208065, 6-(butylsulfinyl)-2,4-diphenylthieno[2,3-d]pyrimidin-5-amine.To the solution of4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile(0.07 mmol, 30 mg) in DMF (0.25 M) was added KOH (0.035 mmol, 2 mg, 0.5equiv., 0.1 M in water). The reaction mixture was stirred at roomtemperature for 20 min. Once complete, the reaction was diluted withEtOAc and washed with 5% aq. solution of acidic acid. The organic phasewas separated and aqueous layer was extracted twice with EtOAc, driedover magnesium sulfate, filtered and concentrated under reduced pressureto give crude product, which was purified by flash chromatography in 70%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.73-8.37 (m, 2H), 7.78-7.68(m, 2H), 7.66-7.55 (m, 3H), 7.53-7.40 (m, 3H), 4.83 (s, 2H), 3.30 (ddd,J=12.7, 8.9, 6.3 Hz, 1H), 3.21-3.01 (m, 1H), 1.87-1.66 (m, 2H),1.57-1.41 (m, 2H), 0.95 (t, J=7.3 Hz, 3H). EST-MS (m/z): 408 [M+H]⁺

4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile.Acetic Acid (600 μL) and hydrogen peroxide (0.37 mmol, 1.5 equiv., 30%solution in water) were added to the solution of4-(((butylthio)methyl)thio)-2,6-diphenylpyrimidine-5-carbonitrile (0.25mmol, 98 mg) in chloroform (900 μL). The reaction mixture was stirringat 32° C. for 45 min. Once complete, the reaction was diluted with EtOAcand washed with saturated NaHCO₃ solution, dried over magnesium sulfate,filtered and concentrated under reduced pressure to give 88 mg ofdesigned product (98%). ¹H NMR (400 MHz, CDCl₃) δ 8.57 (dt, J=7.7, 1.2Hz, 2H), 8.28-8.05 (m, 2H), 7.80-7.40 (m, 6H), 4.82 (d, J=13.2 Hz, 1H),4.49 (d, J=13.3, 1H), 2.95 (dt, J=13.0, 8.1 Hz, 1H), 2.84 (dt, J=13.0,7.3 Hz, 1H), 1.91-1.74 (m, 2H), 1.56-1.40 (m, 2H), 0.95 (t, J=7.4 Hz,3H). ESI-MS (m/z): 408 [M+H]⁺

4-(((butylthio)methyl)thio)-2,6-diphenylpyrimidine-5-carbonitrile. Amixture of 4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.35 mmol, 101 mg), butyl(chloromethyl)sulfane (0.35 mmol, 48 mg, 1.0equiv.) and Et₃N (0.87 mmol, 2.5 equiv.) was refluxed in dry CH₃CN (200μL) for 20 min. The reaction was diluted with EtOAc and water. Theorganic phase was separated and aqueous layer was extracted twice withEtOAc. The combined extractions were washed with saturated NaClsolution, dried over magnesium sulfate, filtered and concentrated underreduced pressure. The residue obtained was then purified by flashchromatography to give 59 mg of designed product (75%). ¹H NMR (400 MHz,CDCl₃) δ 8.75-8.36 (m, 2H), 8.35-7.91 (m, 2H), 7.71-7.41 (m, 6H), 4.59(s, 2H), 2.74 (t, J=7.5 Hz, 2H), 1.75-1.58 (m, 2H), 1.49-1.34 (m, 2H),0.91 (t, J=7.3 Hz, 3H). EST-MS (m/z): 392 [M+H]⁺.

4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile was preparedaccording procedure described by Soto. A mixture of NaOiPr (1.5 mmol,1.0 equiv., prepared in situ from sodium and dry iPrOH), benzothioamide(1.5 mmol, 205 mg, 1.0 equiv.) and2-(ethoxy(phenyl)methylene)malononitrile (1.5 mmol, 297 mg, 1.5 equiv.)in iPrOH (75 mL) was stirred for 5 h at room temperature. The reactionwas then acidified with con. HCl and stirred overnight, evaporated andobtained solid was recrystallized from acetic acid to give 265 mg of4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (61%). ¹H NMR(400 MHz, DMSO-d₆) δ 8.23-8.12 (m, 2H), 8.07-7.91 (m, 2H), 7.74-7.49 (m,6H). ESI-MS (m/z): 290 [M+H]⁺

SW208067,6-(butylsulfinyl)-4-phenyl-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-5-aminewas prepared by using synthetic procedures described for the preparationof analog SW208065 and showed in Scheme 3. ¹H NMR (400 MHz, CDCl₃) δ8.10 (dd, J=3.7, 1.3 Hz, 1H), 7.74-7.65 (m, 2H), 7.62-7.53 (m, 3H), 7.50(dd, J=5.0, 1.2 Hz, 1H), 7.14 (dd, J=5.0, 3.7 Hz, 1H), 4.79 (s, 2H),3.28 (ddd, J=12.8, 9.0, 6.2 Hz, 1H), 3.11 (ddd, J=12.8, 9.0, 6.7 Hz,1H), 1.84-1.63 (m, 2H), 1.54-1.41 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 414 [M+H]⁺.

SW206976-1,1-(3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)pentan-1-one.To the solution of2-((2-oxohexyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile (0.13mmol, 50 mg) in ethanol (500 μL) was added KOH (0.13 mmol, 2 mg, 1.0equiv.). The reaction mixture was stirred at 50° C. for 30 min. Oncecomplete, the reaction was diluted with EtOAc and washed with 10% aq.HCl. The organic phase was separated and aqueous layer was extractedtwice with EtOAc, dried over magnesium sulfate, filtered andconcentrated under reduced pressure to afford designed product in 98%yield.

¹H NMR (400 MHz, CDCl₃) δ 7.65 (dd, J=3.8, 1.1 Hz, 1H), 7.62-7.56 (m,2H), 7.55-7.48 (m, 4H), 7.40 (s, 1H), 7.13 (dd, J=5.0, 3.8 Hz, 1H), 4.13(s, 2H), 2.72 (t, J=7.4 Hz, 2H), 1.72-1.56 (m, 2H), 1.42-1.25 (m, 2H),0.88 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 393 [M+H]⁺.

2-((2-oxohexyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile. Amixture of4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.068 mmol, 20 mg), Et₃N (0.11 mmol, 15 μL, 1.6 equiv.) and2-butyloxirane (0.11 mmol, 11 mg, 1.6 equiv.) in MeOH (500 μL) wasstirred at room temperature. When the reaction was complete as judged byTLC, the reaction mixture was evaporated; the crud product dissolved inDCM and DMP (0.10 mmol, 1.5 equiv.) was added at 0° C. The reactionmixture was stirred at room temperature for 2 h and then was quenched byaddition of 1:1 mixture of 20% Na₂S₂O₃/NaHCO₃ solution. The organiclayer was separated, dried over magnesium sulfate and the solvent wasremoved under reduced pressure. The crude product was purified by flashchromatography to afford designed product in 72% yield. ¹H NMR (400 MHz,CDCl₃) δ 8.02 (s, 1H), 7.97 (d, J=3.1 Hz, 1H), 7.71-7.59 (m, 2H), 7.55(d, J=3.2 Hz, 1H), 7.55-7.46 (m, 4H), 4.52 (s, 2H), 2.75 (t, J=7.8 Hz,2H), 1.73-1.54 (m, 2H), 1.51-1.26 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 393 [M+H]⁺.

SW206977,3-amino-4-phenyl-N-propyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxamide.A mixture of4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.12 mmol, 35 mg), 2-chloro-N-propylacetamide (0.12 mmol, 16 mg, 1.0equiv.) and EtONa (0.19 mmol, 1.6 equiv.) in ethanol (1 mL) was stirredat 50° C. When the reaction was complete as judged by TLC, the reactionwas diluted with EtOAc and washed with 10% aq. HCl. The organic phasewas separated and aqueous layer was extracted twice with EtOAc, driedover magnesium sulfate, filtered and concentrated under reduced pressureto afford designed product in 61% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.65(d, J=3.7, 1H), 7.58-7.49 (m, 3H), 7.49-7.38 (m, 4H), 7.10 (dd J=4.9,3.7 Hz, 1H), 5.75 (s, 2H), 5.59-5.38 (m, 1H), 3.35 (td J=7.0, 5.9 Hz,1H), 1.64-1.58 (m, 2H), 0.96 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 394[M+H]⁺.

SW206978, Ethyl3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxylate. Amixture of4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.34 mmol, 100 mg), ethyl 2-chloroacetate (0.54 mmol, 1.6 equiv.) andEtONa (0.54 mmol, 1.6 equiv.) in ethanol (1 mL) was stirred at reflux.When the reaction was complete as judged by TLC, the reaction wasdiluted with EtOAc and washed with 10% aq. HCl. The organic phase wasseparated and aqueous layer was extracted twice with EtOAc, dried overmagnesium sulfate, filtered and concentrated under reduced pressure toafford designed product in 79% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.01(d, J=3.7 Hz, 1H), 7.87-7.67 (m, 2H), 7.56 (d, J=6.5 Hz, 5H), 7.36-6.90(m, 1H), 5.73 (s, 2H), 4.23 (q, J=7.1 Hz, 2H), 1.25 (t, J=7.0 Hz, 3H).ESI-MS (m/z): 381[M+H]⁺.

SW206979,3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxylicacid. To a solution of-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(0.34 mmol, 100 mg) and ethyl 2-chloroacetate (0.54 mmol, 1.6 equiv.) inethanol (1 mL), Et₃N (0.54 mmol, 1.6 equiv.) was added. The reaction wasrefluxed for 20 min. The reaction was then diluted with EtOAc and water.The organic phase was separated and aqueous layer was extracted twicewith EtOAc. The combined extractions were washed with saturated NaClsolution, dried over magnesium sulfate, filtered and concentrated underreduced pressure to afford designed product. Ethyl2-((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)acetate was thendissolved in DMF and treated with 1M aq. NaOH at 50° C. to giveSW206979,3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxylicacid in 63% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (dd, J=3.7, 1.1 Hz,1H), 7.79-7.64 (m, 2H), 7.55 (dt, J=7.6, 3.2 Hz, 5H), 7.16 (dd, J=5.0,3.7 Hz, 1H), 5.72 (s, 2H). ESI-MS (m/z): 353[M+H]⁺.

SW208068, 2-(butylthio)pyridin-3-amine. To the solution ofbutane-1-thiol (7.0 mmol, 628 mg, 1.1 equiv.) in THF (30 mL) was addedNaH (6.6 mmol, 158 ng, 1.05 equiv.) at 0° C. After the reaction mixturewas stirred at room temperature for 30 min. 2-chloro-3-nitropyridine(6.33 mmol, 1.0 g) was portion wise added and left with stirring at roomtemperature. for 2 h. Water was then added to the reaction mixture, andthe resulting mixture was extracted with ethyl acetate. The organiclayer was washed with a saturated aqueous solution of sodium chloride,and dried over sodium sulfate, filtered and concentrated under reducedpressure to afford crude product. Because of difficulties withpurification, impure 2-(butylthio)-3-nitropyridine was directly used forthe next step. Nitropyridine (0.47 mmol, 100 mg) was dissolved in amixed solvent of acetic acid (3.3 ml) and conc. hydrochloric acid (130μL), and zinc (5.7 mmol, 370 mg) was added in small portions while beingcooled with ice. After the mixture was stirred for 30 minutes, thereaction mixture was filtered, and the filtrate was neutralized with anaqueous solution of NaHCO₃, and extracted with DCM. The organic layerwas washed with water and then with a saturated aqueous solution ofsodium chloride, and dried over sodium sulfate. Subsequently, thesolvent was evaporated to obtain 2-(butylthio)pyridin-3-amine as a paleyellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (dd, J=4.1, 2.0 Hz, 1H),7.05-6.51 (m, 2H), 3.84 (s, 2H), 3.51-2.95 (m, 2H), 1.72-1.60 (m, 2H),1.56-1.36 (m, 2H), 0.91 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 183 [M+H]⁺.

SW208069, 2-(butylsulfinyl)-3-nitropyridine. To the solution ofbutane-1-thiol (7.0 mmol, 628 mg, 1.1 equiv.) in THF (30 ml.) was addedNaH (6.6 mmol, 158 mg, 1.05 equiv.) at 0° C. After the reaction mixturewas stirred at room temperature for 30 min. 2-chloro-3-nitropyridine(6.33 mmol, 1.0 g) was portion wise added and left with stirring at roomtemperature. for 2 h. Water was then added to the reaction mixture, andthe resulting mixture was extracted with ethyl acetate. The organiclayer was washed with a saturated aqueous solution of sodium chloride,and dried over magnesium sulfate, filtered and concentrated underreduced pressure to afford crude product. Because of difficulties withpurification, impure 2-(butylthio)-3-nitropyridine was used directly forthe next step. Nitropyridine (0.47 mmol, 100 mg) was dissolved in amixed solvent of acetic acid (1.2 ml) and chloroform (1.2 mL), andhydrogen peroxide (0.7 mmol, 1.5 equiv., 30% solution in water) wasadded. After the mixture was stirred for 45 minutes, at 32° C., thereaction was diluted with EtOAc and washed with saturated NaHCO₃solution, dried over magnesium sulfate, filtered and concentrated underreduced pressure to 2-(butylsulfinyl)-3-nitropyridine. ¹H NMR (400 MHz,CDCl₃) δ 9.14 (dd, J=4.6, 1.5 Hz, 1H), 8.54 (dd, J=8.2, 1.5 Hz, 1H),7.67 (dd, J=8.2, 4.6 Hz, 1H), 3.18 (ddd, J=12.7, 9.3, 7.2 Hz, 1H), 3.00(ddd, J=12.7, 9.1, 4.9 Hz, 1H), 2.17-1.92 (m, 1H), 1.91-1.70 (m, 1H),1.68-1.35 (m, 2H), 0.96 (t, J=7.3 Hz, 3H). EST-MS (m/z): 229 [M+H]⁺.

SW208070, 2-(butylsulfinyl)pyridin-3-amine was prepared by usingsynthetic procedure described for the preparation of analog SW208069 andshowed in Scheme 4. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J=4.4, 1.4 Hz,1H), 7.09 (dd, J=8.3, 4.4 Hz, 1H), 6.93 (dd, J=8.3, 1.4 Hz, 1H), 5.30(s, 2H), 3.24 (ddd, J=13.0, 9.5, 5.4 Hz, 1H), 3.03 (ddd, J=13.0, 9.8,6.3 Hz, 1H), 1.95-1.61 (m, 2H), 1.55-1.35 (m, 2H), 0.93 (t, J=7.3 Hz,3H). ESI-MS (m/z): 199 [M+H]⁺.

Example 4 Analysis of the Mechanism of 15-PGDH Inhibition by SW033291and Related Compounds

The following Example provides data relating to the mechanism of actionby which SW033291 inhibits 15-PGDH.

Duplicate titrations of 15-PGDH Inhibitor (SW033291) were run at 4different concentrations of 15-PGDH(24 nM, 12 nM, 6 nM, 3 nM). Reactionscontained the indicated concentration of enzyme, 250 μM NAD(+), 25 μMPGE-2, and were assembled and incubated at room temperature for 3minutes.

FIG. 21 (A-B) show the shift in IC₅₀ value with changing enzymeconcentration. The result is indicative of a tight-binding mode ofinhibition with dependency on enzyme: inhibitor stoichiometry, ratherthan on the absolute concentration of the inhibitor. In all cases, theIC₅₀ values are less than the enzyme concentration, indicating that nMdrug is almost fully bound by the enzyme.

FIG. 22 (A-B) indicate that SW033291 behaves very much like anirreversible inhibitor of 15-PGDH, and cannot be efficiently dialyzedoff the 15-PGDH protein.

The testing of whether SW033291 is a reversible inhibitor proceeded by:

-   -   (i) an 8 ul aliquot taken of 15-PGDH stock (8 mg/mL 15-PGDH in        500 μL of 15-PGDH assay buffer)(4 nmol 15-PGDH, 4 μM 15-PGDH),        was incubated on ice with: (a) addition of 5 μL of 100 mM        NAD(+)+addition of 3.2 μL of 2.5 mM SW033291 stock, then        dialyzed versus 1 L buffer for 12 hours, followed by a fresh 1 L        of buffer for 12 more hours; or (b) addition of 5 μL, 100 mM        NAD(+)+addition of 3.2 μL DMSO then dialyzed versus 1 L buffer        for 12 hours, followed by a fresh 1 L of buffer for 12 more        hours.    -   (ii) Pre-dialysis, remove 1 μL and dilute in 200 μL assay Buffer        (20 nM), then measure 15-PGDH activity.    -   (iii) Post-Dialysis, at 24 hrs, remove 1 μL and dilute in 200 μL        assay Buffer (20 nM), then measure 15-PGDH activity.

Dialysis buffer is 50 mM Tris pH7.4, 40 mM NaCl, 0.1 mM DTT, 0.01%Tween-20.

Under the conditions of the assay SW033291 inhibited 91% of 15-PGDHpre-dialysis, and 85% of 15-PGDH activity post-dialysis—that is dialysisdid not reverse the inhibition of 15-PGDH. Control 15-PGDH protein thatwas dialyzed in the absence of SW033291 remained fully active.

FIGS. 23 (A-B) show (A) at upper right the reaction rates for 15-PGDH inthe presence of a graded set of increasing concentrations of SW033291.In the graph at upper right P is the NADH concentration as a proxy for15-keto-PGE₂. (P+S) is the starting PGE₂ concentration of 20 μM. Theassay was carried out in the presence of 10 nM recombinant 15-PGDH. Inthe graph at lower left (B), Vo is the initial velocity of the reactionin the absence of SW033291, and Vi is the initial velocity of thereaction in the presence of the corresponding concentration of SW033291.The line shows the curve generated by fitting the data to the Morrisonequation. The curve fitting yields a calculated a Ki^(App) value of0.1015 nM. The dashed line intersects the X axis at 8.5 nM. Thisrepresents the point at which [inhibitor]=[active enzyme] showing thatthe enzyme preparation contains 85% active enzyme. In the Morrisonequation, Ki is the binding affinity of the inhibitor; [S] is substrateconcentration; and Km is the concentration of substrate at which enzymeactivity is at half maximal. Note that IC50 is the functional strengthof the inhibitor. Whereas the IC50 value for a compound may vary betweenexperiments depending on experimental conditions, the Ki is an absolutevalue. Ki is the inhibition constant for a drug; the concentration ofcompeting ligand in a competition assay which would occupy 50% of thereceptors if no ligand were present.

FIGS. 24 (A-B) show duplicate titrations of 15-PGDH Inhibitor (SW033291)that were run at 6 different concentrations of PGE₂ (1.25 uM-40 uM). Inthe graph at top, Y-axis is % inhibition of the reaction by SW033291.The X-axis is the concentration of SW033291 in nM. Reactions contain 5.0nM added 15-PGDH, 250 μM NAD(+), and indicated concentrations of PGE-2,were assembled and incubated at room temperature for 60 minutes. The Kmfor PGE₂ is approximately 5 μM, and reactions run with PGE₂concentrations below 5 uM go very slowly making it difficult toquantitate inhibition by SW033291. However, in reactions with PGE₂ atconcentrations of 5 uM-40 μM, the IC₅₀ for SW033291 is unaffected by theincreasing PGE₂ concentration, showing that the inhibition isnoncompetitive.

FIG. 25 shows the structure activity relationships of analogues ofSW033291 versus their IC50 against recombinant 15-PGDH. Assignments ofstructures to the two isomers, A and B, of SW033291 are arbitrary, asthe structure of the active isomer (isomer B) has not been determined.The optical isomers of SW033291 were separated by preparative HPLC usinga 10 mm×250 mm Chiralcel ODH column, 5% isopropanol in hexanes, 1mL/min. The ‘A’ isomer is the faster eluting isomer. The ‘B’ isomer isthe slower eluting isomer.

The analogue family shows that SW033291, with a 4 carbon side chain, is2-fold more potent than SW208080 (5 carbon side chain), 15-fold morepotent than SW208081 (6 carbon side chain), and 100-fold more potentthan SW208079 (1 carbon side chain). SW033291 is also 20-fold morepotent than SW208078, the analogue that converts the sulfoxide group toa sulfone.

FIG. 26 shows structures of additional SW033291 analogs that convert thesulfoxide group to a ketone, an amide, an ester, or a carboxylic acid.Also shown is structure SW206980 that deletes the phenyl ring fromSW033291.

FIGS. 27 (A-C) show graphs that show the level of compound's activity ininducing the 15-PGDH-luciferase fusion reporter in three different testcell line backgrounds, V9m, LS174T, and V503. Each compound was testedat two concentrations, 2.5 uM, and 7.5 uM. Y-axis is luciferaseactivity.

At 2.5 μM-7.5 μM, SW206980, that deletes the phenyl group of SW033291,shows activity comparable to SW033291 in all three reporter lines.

Structures that have converted the sulfoxide group to a ketone, amide,ester, or carboxylic acid show major loss of activity in inducing thereporter.

FIG. 28 shows graphs that show the percent of 15-PGDH enzyme activitythat is inhibited at 2.5 uM and at 7.5 uM by each of the 5 testcompounds. SW206980 that deletes the phenyl group of SW033291, shows atthese concentrations similar potency to SW03291 in inhibiting 15-PGDHactivity.

Structures that have converted the sulfoxide group to a ketone, amide,ester, or carboxylic acid show major loss of activity as 15-PGDHinhibitors.

FIGS. 29 (A-B) show a titration curve that plots percent inhibition of15-PGDH enzyme activity at different concentrations of SW033291 andSW0206980. Under identical assay conditions, SW206980 shows a slightlylower IC50.

FIGS. 30 (A-B) show that SW206980 binds directly to 15-PGDH and markedlyshifts its melting curve. Shown at left is the melt curve of 15-PGDH asreflected by fluorescence of the hydrophobic dye SYPRO Orange. Shown atright is the negative first derivative of the melt curve.

Three conditions are plotted, that of 10 uM 15-PGDH, that of 10 uM15-PGDH plus 10 uM SW206980, and that of 10 uM 15-PGDH plus 125 μM NADHplus 10 μM SW206980. The melting temperature, as reflected by theinflection point of the curve at right is shifted by 20° C., from48-degrees up to 68-degrees, in the presence of SW206980 and NADH,reflecting that SW206980 directly binds to and markedly stabilizes thetertiary structure of 15-PGDH, in a manner requiring the presence of theNADH cofactor.

FIGS. 31 (A-C) show further analogues of SW033291 that build on theprevious finding that removal of the SW033291 phenyl ring (SW206980)retained activity. The new analog (SW206992) adds a nitrogen to theleft-hand ring.

Table 3 provides a comparison of the properties of SW033291, SW206980,and SW206992.

TABLE 3 Summary of three SW033291 analogs SW033291 SW206980 SW206992IC₅₀  1.59 nM  0.97 nM 1.411 nM Time to inhibition ~5 mins ~2 mins ~2mins (10 nM) Δ Tm (NADH) 19° C. 15.5° C. 19° C. Concentration for ~100nM >300 nM >1 uM Full Cell Line Reporter Induction Hepatocyte stabilityStable > couple hrs T½ = 80 mins Toxicity >10 μM >7.5 μM >7.5 μM

Time to inhibition refers to the time needed to inhibit the generationof NADH by 15-PGDH from the moment with drug is added into the reactionmix. Delta Tm refers to the shift in melting temperature of recombinant15-PGDH in the presence of drug (with cofactor NADH also present).Concentration of Full Cell Line Reporter Induction refers to theconcentration of drug that needs to be added to reporter cell line toachieve maximal induction of the 15-PGDH-luciferase gene fusion reportercassette, as measured by luciferase assays. Hepatocyte stability refersto the half-life of compound in the presence of hepatocytes in culture.Toxicity refers to the concentration of compound needed to decrease cellnumbers in a cell culture assay.

FIGS. 32 (A-C) show titration of induction by SW033291 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general between 80-160 nM SW033291 exposure for 24 hoursis needed to induce maximal reporter induction.

FIGS. 33 (A-C) show titration of induction by SW206980 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general ≥300 nM SW206980 exposure for 24 hours is neededto induce maximal reporter induction.

FIGS. 34 (A-C) show titration of induction by SW206992 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general ≥1000 nM SW206992 exposure for 24 hours isneeded to induce maximal reporter induction.

FIGS. 35 (A-C) show the shift in the melt curve of recombinant 15-PGDHprotein (10 uM) by 20 uM SW033291, SW206980, and SW206992 in thepresence of the cofactor NAD (+)(100 uM). Control melting temperature is49 degrees centigrade. SW033291 shifts the melting temperature to 63degrees. SW206980 shifts the melting temperature to 61 degrees. SW206992shifts the melting temperature to 59 degrees. Thus all three compoundsdirectly bind to 15-PGDH and markedly increase the melting temperatureof the protein, with the order of the temperature shifts beingSW033291>SW206980>SW206992.

FIGS. 36 (A-B) show the shift in the melt curve of recombinant 15-PGDHprotein (10 uM) by 20 uM SW033291, SW206980, and SW206992 in thepresence of the cofactor NADH (100 uM). Control melting temperature is55 degrees centigrade. SW033291 shifts the melting temperature to 74degrees. SW206980 shifts the melting temperature to 70.5 degrees.SW206992 shifts the melting temperature to 68.5 degrees. Thus all threecompounds directly bind to 15-PGDH and markedly increase the meltingtemperature of the protein, with the order of the temperature shiftsbeing SW033291>SW206980>SW206992.

FIGS. 37 (A-C) show a titration curve of 15-PGDH inhibitor compounds inan assay measuring effect on PGE₂ levels in the medium of A549 cellsthat have been stimulated with IL1-beta. Highest PGE₂ levels, 3000pg/ml, are achieved with SW033291, with maximal effect attained at 2.5uM compound. Next highest PGE₂ level, 2500 pg/ml are achieved withSW206980, with maximal effect attained at 7.5 uM compound. Lowestinduction, to 2100 pg/ml PGE₂ is achieved with SW206992, with maximaleffect attained at 2.5 uM. In these reactions, A549 cells weremaintained in F12K medium supplemented with 10% fetal calf serum (FBS)and 50 pg/mL gentamicin in a humidified atmosphere containing 5% CO₂ at37° C. Cells were plated in 24-well plates (0.5 mL per well) at about100,000 cells per well in duplicate and grown for 24 h beforestimulation with IL-1β (1 ng/mL) overnight (16 h) to generate PGE₂.SW033291 and its analogs were added at the indicated concentrations, andthe incubation continued for 8 h. Medium was collected, and the level ofPGE₂ was analyzed by enzyme immunoassay. Data were analyzed from resultsof three independent experiments.

FIGS. 38 (A-C) show assays of cellular toxicity on A549 cells at 24hours of 15-PGDH inhibitors as assayed by CellTiter-Glo measurement. Noeffect on CellTitre-Glo levels is seen by concentrations of up to 10 uMof SW033291, SW206980, and SW2206992.

FIG. 39 shows structures of 7 SW033291 analogues, SW208064, SW208065,SW208066, SW208067, SW208068, SW208069, SW208070.

Table 4 provides tabular summary of the properties of 4 analogues,SW208064, SW208065, SW208066, SW208067, and in particular lists the IC50for each of these 4 compounds against 2.5 nM recombinant 15-PGDH.

TABLE 4 Summary of four SW033291 analogs from UTSW set 8 SW033291SW2068064 SW208065 SW208066 SW208067 IC₅₀  1.23 nM 151.4 nM 4.865 nM1.368 nM 2.395 nM Time to ~5 mins inhibition (10 nM) Δ Tm 19° C. 5° C.13° C. 16.5° C. 16.5° C. (NADH) Concen- ~100 nM ~600 nM  ~100 nM ~100 nM~500 nM tration for Full Cell Line Reporter Induction HepatocyteStable > stability couple hrs Toxicity >10 μM

Time to inhibition refers to the time needed to inhibit the generationof NADH by 15-PGDH from the moment with drug is added into the reactionmix. Delta Tm refers to the shift in melting temperature of recombinant15-PGDH in the presence of drug (with cofactor NADH also present).Concentration of Full Cell Line Reporter Induction refers to theconcentration of drug that needs to be added to reporter cell line toachieve maximal induction of the 15-PGDH-luciferase gene fusion reportercassette, as measured by luciferase assays. Hepatocyte stability refersto the half-life of compound in the presence of hepatocytes in culture.Toxicity refers to the concentration of compound needed to decrease cellnumbers in a cell culture assay.

FIG. 40 provides graphical summary showing the activity of each of thecompounds in inducing a 15-PGDH-luciferase fusion gene reporterintroduced into three different colon cancer cell lines, V9m, LS174T,and V503. Results are measured by assay of luciferase activity afterexposure of cells to compound at either 2.5 uM or 7.5 uM compoundsconcentration.

FIG. 41 provides graphical summary showing the activity of each of thecompounds in inhibiting the enzymatic activity of recombinant 15-PGDHenzyme when compound is added at 2.5 uM and at 7.5 uM. 100% inhibitioncorresponds to complete inhibition of the enzyme.

FIG. 42 shows measurement of IC for inhibiting 2.5 nM of recombinant15-PGDH when incubated across a range of concentrations of SW208064,SW208065, SW208066, and SW208067. Y-axis of each graph records percentinhibition of the 15-PGDH enzymatic activity. 100% Inhibitioncorresponds to complete inhibition of the enzyme. X-axis of each graphrecords the log of the inhibitor concentration expressed in nM.

Example 5 Analysis of Toxicity of SW033291

Table 5 shows a summary of a group of 8-12 week old male FVB mice incontrol or SW033291 treatment arms assessed for toxicity of SW033291,with 6 mice in each arm of the study.

TABLE 5 Baseline Characteristics FVB male mice- 8-12 weeks old ToxicityStudy WT-Control WT-Treatment p-value Number 6 6 Sex M M Age (Days)73.7.1 ± 4.7 73.2 ± 5.0 0.465 Weight (gm)   27.5 ± 2.4 26.8 ± 3.1 0.412

FIG. 43 shows the dose response curve for induction of a15-PGDH-luciferase fusion gene reporter in the V9m cell line backgroundof SW033291, SW208064, SW208065, SW208066, and SW208067.

FIG. 44 Shows titration curves of 15-PGDH inhibitor compounds in anassay measuring effects on PGE₂ levels in the medium of A549 cells thathave been stimulated with IL1-beta in the same experimental designdescribed for FIG. 37 . At 100 nM concentration of drug, the highestlevels of PGE₂ in the medium are achieved by treating cells withSW208066 or with SW208067, after which the next highest level of PGE₂ inthe medium is achieved by treating cells with SW033291.

FIG. 45 shows the daily weights of a group of 8-12 week old FVB micetreated with vehicle or with SW033291 IP at 5 mg/kg twice daily for 21days. SW033291 was administered in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W at a concentration of 125 ug/200 ul. As shown,both vehicle and drug treated mice show equal weight gain during the 21day period, with no evidence for SW033291 reducing mouse weight. N=6mice in both the SW033291 treated and the vehicle treated arms.

Example 6 Analysis of Effect of SW033291 on Bone Marrow Function

This Example shows effects of SW033291 on bone marrow function.

FIGS. 46 (A-C) show analysis of bone marrow of wild-type mice versusmice that are homozygous genetic knockouts for 15-PGDH (PGDH −/− mice).Total bone marrow cellularity and percent of Scal+/c-Kit+ cells inlineage negative (SKL) cells are the same in both sets of mice. However,bone marrow from 15-PGDH −/− mice shows an approximately 50% increase innumbers of hematopoietic colonies generated when marrow is plated intomethylcelluose. Experimental conditions are noted on the figure. 15-PGDHknockout mice are denoted by label PGDH −/− and by label 15-PGDH.

FIG. 47 shows assay in which bone marrow is harvested from a wild-typemouse, and incubated ex vivo on ice for 2 hours with either SW033291(0.5 uM), or 1 uM PGE₂ or 1 uM 16,16-dimethyl PGE₂ (dmPGE2). Treatedmarrow is again then plated into methylcellulose for counting ofhematopoietic colonies. SW033291 treated marrow again shows anapproximately 50% increase in the number of bone marrow derived coloniesgenerated. Under these conditions, a lesser increase is seen in marrowtreated with PGE₂, and a slightly greater increase is seen in marrowtreated with dmPGE2.

FIGS. 48 (A-C) show a study of C57BL/6J mice treated with IP SW033291administered in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W at adose of 5 mg/kg or 20 mg/kg. Panel A shows mouse bone marrowcellularity, white blood count (wbc), red blood count (rbc) andplatelets counts. Panel B shows percent of Scal+/c-Kit+ cells in lineagenegative (SKL) cells are unchanged in SW033291 treated mice. Panel Cshows that marrow from SW033291 treated mice gives rise to approximately30% increase in numbers of hematopoietic colonies generated when marrowis plated into methylcelluose. Experimental conditions are noted on thefigure.

FIGS. 49 (A-B) show analysis of marrow from CD45.2 antigen markedC57BL/6J mice that were treated with SW033291 5 mg/kg IP daily for 3doses in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W or that weretreated with vehicle alone. On day 3 mice were sacrificed, marrowflushed and mixed at a 1:1 ratio with vehicle treated CD45.1 marrow. 2million whole BM cells were injected into the tail vein of lethallyirradiated CD45.1 mice and percent chimerism measured via flow cytometryat weeks 8, 12, 16. As shown, at weeks 12 and 16 the percent bloodchimerism of CD45.2 marked cells was significantly increased inrecipient mice whose CD45.2 marked marrow was harvested from SW033291treated donor mice, as opposed to vehicle control treated donor mice. Inother words, marrow from SW033291 treated mice demonstrated long termincreased fitness in competition with control marrow. In particular, atweek 16 CD45.2 harvested from SW033291 treated mice show a significantincrease in contribution to B and T cell populations, suggesting marrowfrom SW033291 treated mice promotes earlier reconstitution of lymphoidpopulations and earlier return to immune competence.

In an additional study, C57BL/6J mice are irradiated with 11Gy on day 0,followed by treatment with SW033291 5 mg/kg IP twice daily (bid) in avehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W, or with vehicle onlyfor 21 days. Mice treated with vehicle or with SW033291 all receive anallograft of marrow from a donor C57BL/6J mouse at a dose of either100,000 cells, 200,000 cells, 500,000 cells. 3 control and 3 SW033291mice are assessed under each condition. The experimental design isdepicted in FIG. 50 .

Table 6 shows the number of surviving mice in each cohort over the first19 days of study. Under the conditions of the mouse colony during thisstudy, control mice receiving 100,000-500,000 cells are all dead betweendays 4-13 of study. In contrast, two SW033291 treated mice receiving500,000 cells remain alive on day 19 of the study and are presumed tohave full hematopoietic reconstitution. Thus treatment with the 15-PGDHinhibitor SW033291 promoted survival of mice receiving a bone marrowtransplant, an observation consistent with SW033291 enabling more rapidand complete hematopoietic reconstitution in the transplanted mice.Other 15-PGDH inhibitors with activity similar to SW033291 would bepredicted to have similar activity in supporting hematopoieticreconstitution. Treatment with SW033291 also enabled mice to besuccessfully transplanted with a smaller inoculum of donor bone marrowthan the 1,000,000 cells that are standardly needed. These observationssuggest SW033291, as well as other similar 15-PGDH inhibitors, is ableto support successful transplantation with smaller numbers of donor stemcells. Such activity would be of particular utility in settings, such astransplantation with umbilical cord stem cells, in which donor cellnumbers are limited. Improved survival of transplanted mice treated withSW033291 suggests efficacy of SW033291, and of similar 15-PGDHinhibitors, as replacements for, or in enabling decreased use of, othertreatments or growth factors commonly employed in support of patientsreceiving bone marrow, hematopoietic stem cell, and cord blood stem celltransplants. Improved survival of transplanted mice treated withSW033291 is consistent with SW033291, and by extension other similar15-PGDH inhibitors, having activity in reducing infections in thetransplanted mice, and/or in promoting recovery of mice intestines fromdamage by radiation, and/or in reducing pulmonary toxicity fromradiation.

TABLE 6 Mouse 13 14 15 16 17 18 19 20 21 survival March March MarchMarch March March March March March Cell Day Day Day Day Day Day Day DayDay number 0 1 2 3 4 5 6 7 8 Control 1 × 10{circumflex over ( )}5 3 3 32 0 Control 2 × 10{circumflex over ( )}5 3 3 3 3 3 2 1 0 Control 5 ×10{circumflex over ( )}5 3 3 3 3 3 3 2 1 1 SW033291 1 × 10{circumflexover ( )}5 3 3 3 3 2 1 0 SW033291 2 × 10{circumflex over ( )}5 3 3 3 3 33 2 2 2 SW033291 5 × 10{circumflex over ( )}5 3 3 3 3 3 3 3 3 2 Mouse 2223 24 25 26 1 Survival March March March March March . . . April Day DayDay Day Day Day Treatment Cell number 9 10 11 12 13 19 Control 1 ×10{circumflex over ( )}5 Control 2 × 10{circumflex over ( )}5 Control 5× 10{circumflex over ( )}5 1 1 1 1 0 SW033291 1 × 10{circumflex over( )}5 SW033291 2 × 10{circumflex over ( )}5 2 2 2 1 0 SW033291 5 ×10{circumflex over ( )}5 2 2 2 2 2 . . . 2

Example 7 Analysis of Effect of SW033291 on Radiation Survival

This Example shows studies of the effect of SW033291 in mice receivingwhole body irradiation.

Table 7 shows the results of a study of 15 week old C57BL/6J female miceirradiated with 7Gy, 9Gy, or 11Gy, and receiving daily SW033291 5 mg/kgIP in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W for 7 doses, orreceiving vehicle alone. The table shows the number of mice surviving onsequential days of the study. Under the conditions of the mouse colonyduring this experiment, mice receiving a lethal dose of 11Gy lived 48hours longer if treated with SW033291 than if receiving vehicle control,with control mice all dead on day 8; whereas SW033219 treated mice wereall dead on day 10.

TABLE 7 October October October October October October October 2 3 4 56 7 8 Radiation Treatment Day Day Day Day Day Day Day Dose Arm 0 5 6 7 89 10  7 Gly Saline 3 3 3 3 3 3 3 SW033291 3 3 3 3 3 3 3  9 Gy Saline 3 33 3 3 3 3 SW033291 3 3 3 3 3 3 3 11 Gy Saline 3 3 3 2 0 SW033291 3 3 3 33 2 0 October October October October October October 9 10 11 12 13 23Radiation Treatment Day Day Day Day Day Day Day Dose Arm 11 12 13 14 1516 25  7 Gly Saline 3 3 3 3 3 Looks Healthy SW033291 3 3 3 3 3 LooksHealthy  9 Gy Saline 3 2 1 1 0 SW033291 3 2 2 1 0 11 Gy Saline SW033291

Table 8 shows the number of mice surviving on sequential days of a studyof mice treated at 11Gy treated with either vehicle control or withSW033291 IP, in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W, withSW033291 administered either at 5 mg/kg daily for 7 days, 5 mg/kg dailythroughout the study, or at 5 mg/kg twice daily for 7 days. Again micetreated with SW033291 on any of these dosing schedules live on average1-2 days longer than mice receiving vehicle control. The activity ofSW033291 in promoting resistance to toxic effects of radiation mayextend to SW033291 and other similar 15-PGDH inhibitors in promotingresistance to other similar toxic insults including but not limited toCytoxan, fludarabine, chemotherapy and immunosuppressive therapy.

TABLE 8 Friday Wed. Thurs. Friday Saturday Sunday Monday 12 17 18 19 2021 22 October October October October October October October TreatmentConditions Day 0 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 11 Gy Saline (7days, 1 does daily) 2 2 2 2 0 11 Gy SW033291 3 3 3 3 2 1 0 (1does/daily) for 7 days 11 Gy SW033291 (1 does/daily, 3 3 3 3 3 2 0continuous every day) 11 Gy Saline (7 days, 2 does/daily 3 3 3 3 1 0 011 Gy SW033291 3 3 3 3 3 2 0 (7 days, 2 dose daily)

Example 8 Analysis of Effect of SW033291 on Liver Regeneration PostPartial Hepatectomy

This Example shows studies assessing the effect of SW033291 on liverregeneration in mice following partial hepatectomy.

FIG. 51 shows a drawing of the anatomy of the mouse liver and of thepartial hepatectomy procedure described in Mitchell et al., NatureProtocols, 3, 1167-1170 (2008), in which the median and left laterallobes are resected, and then liver regeneration is observed viahypertrophy of the remaining right and caudate lobes. The totalresection is of approximately 70% of the mouse liver mass. In thesestudies mice were euthanized using carbon dioxide inhalation. The mousebody was weighed in its entirety. The liver was removed from the mouse;the necrotic remnant from the surgical resection was trimmed; and theregenerated liver was weighed.

FIG. 52 (A-D) show an anatomical view of the mouse liver. Pictures atleft are pre-operative views, and those at right are post-resectionviews. The upper two pictures show anterior view of liver and at leftdisplay the median lobe and a part of the Lateral lobe. The lower twopictures show Inferior view of liver and at left display the Laterallobe. In the partial hepatectomy procedure, the median and lateral lobesare resected as shown at right.

FIG. 53 (A-D) at left reiterates photographs of the immediatepost-hepatectomy views of the mouse liver, with anterior view at top andinferior view at bottom. Figure at top right shows the in situ view ofthe regenerated liver on post operative day (POD) 10, showinghypertrophy of the remnant right and caudate lobes. Photograph at lowerright shows the anterior view of the regenerated liver after removalfrom the mouse body. Whitish region at the upper right liver edge is thenecrotic stump from the resection, and is trimmed prior to weighing.

The first study was performed in 10 week old male C57BL/6J mice,receiving daily SW033291 5 mg/kg IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W, versus vehicle alone, and assessed daily forliver regeneration with 5 control and 5 SW033291 recipient micesacrificed on each time point. In this study, ketamine anesthesia wasemployed.

FIG. 54 (A-B) show micrograph of the hematoxylin and eosin stained liveron post-operative day 3 (POD 3) in the SW033291 treated mouse versus thecontrol mouse, with mitotic figures marked by yellow arrows in theSW033291 treated mouse liver at left and by green arrows in the controlmouse liver at right.

FIG. 55 shows a graph of the number of mitosis per high powered field inlivers of SW033291 treated versus control mice on post-operative daystwo through five (2D-5D). Mitotic figures were counted in 10 highpowered fields (40X) from each of 5 livers per SW033291 or control miceper day. SW033291 treated mice demonstrated significantly increasedhepatic mitosis versus controls on days 3 and 4.

Table 9 shows the numbers of mitosis per random high powered fieldcounted in livers of control versus SW033291 treated mice on postoperative days 2 through 5 (2D-5D). SW033291 treated mice showsignificantly increased numbers of mitotic liver cells on post-operativedays 3 and 4.

TABLE 9 Mitotic index (40×) mean + SD Control SW033291 p-value 2D 0.7000± 0.1933 1.240 ± 0.2330 0.0915 3D  2.000 ± 0.4364 6.160 ± 0.3250 <0.00014D  2.560 ± 0.2242 4.560 ± 0.7190 0.0107 5D 0.2000 ± 0.1069 0.2400 ±0.08718 ns

FIG. 56 shows the liver to body weight ratios attained following partialhepatectomy in control versus SW033291 treated C57Bl/6j mice injected at5 mg/kg SW033291 IP daily (qd) starting on post operative day 0 andcontinuing throughout. Graph displays values from post-operative days2-7 (POD 2-7). The SW033291 qd injection group of mice attain a higherliver to body weight ratio from post-operative days 4-7, with theincrease being statistically significant on postoperative day 4 and day7.

An additional group of mice received SW033291 5 mg/kg twice daily (bid)and were analyzed on post-operative day 3, with the data graphed asPOD3b. This group of mice also showed a statistically significantincrease in liver to body weight ratio compared to control mice.

Another study was performed testing the effects of SW033291 given 5mg/kg IP twice daily (bid) on liver regeneration in C57BL/6J mice. 10mice were used in the control and 10 mice in the drug treated arm foranalysis of each time point of the study. The study again employed 10week old male mice receiving daily SW033291 5 mg/kg IP in a vehicle of10% Ethanol, 5% Cremophor EL, 85% D5W, versus vehicle alone, with 10drug treated and 10 control mice sacrificed daily for comparison. Inthis study ketamine anesthesia was employed.

FIG. 57 shows graph of the liver to body weight ratio attained followingpartial hepatectomy in control versus SW033291 treated C57BL/6J miceinjected at 5 mg/kg SW033291 IP twice daily (bid) starting at 1 hourpost-surgery and continued throughout. Graph displays values frompost-operative days 2-7 (POD 2-7). The SW033291 bid injected group ofmice show a statistically significant higher liver to body weight ratioversus control mice on post-operative days 3, 4, and 7.

FIG. 58 reprises the graph of the liver to body weight ratio attainedfollowing partial hepatectomy in control mice versus in mice treatedwith sw033291 5 mg/kg IP twice daily. In data enclosed within the bluebox, drug was started 1 hour following surgery, and significantincreases in liver to body weight ratio are seen in drug treated micefrom post-operative day (POD) 3 onward. In data enclosed within the redbox, the first dose of sw033291 is delivered commencing 1 hour beforesurgery, and significant increase in liver to body weight ratio is seenas early as post-operative day 1, the day following the surgery.

FIGS. 59 (A-B) show graphs of the serum ALT levels following partialhepatectomy in one control mouse versus one mouse treated with sw033291at 5 mg/kg IP twice daily (bid). Post-operative day 1 values arecompared at left, and post-operative day 2-7 values are compared atright. ALT values are lower in the drug treated mouse.

FIG. 60 shows graph of serum bilirubin levels following partialhepatectomy in one control mouse versus one mouse treated with SW033291at 5 mg/kg IP twice daily (bid) from post-operative days (POD) 1-7.

In another study, SW033291 was tested in the partial hepatectomy modelusing the FVB strain of mice administered SW033291 5 mg/kg IP twicedaily (bid), administered in a vehicle of 10% Ethanol, 5% Cremophor EL,85% D5W, using 5 treated mice versus 5 control mice treated with vehiclealone for analysis at each time point from post operative day (POD) 1-7.In this study ketamine anesthesia was employed.

FIG. 61 shows a graph of the liver to body weight ratio of attainedfollowing partial hepatectomy in FVB mice treated with 5 mg/kg IPSW033291 versus control mice treated with vehicle alone. SW033291treated mice show increased liver to body weight ratio frompost-operative days 2-7, with the increase being statisticallysignificant on POD, 2,3, 4 and 7.

In another study, SW033291 was tested in a partial hepatectomy modelusing the FVB strain of mice administered SW033291 5 mg/kg IP twicedaily (bid), starting 1 hour before surgery. 10 week old male mice wereemployed, with 10 treated mice and 10 control mice used for analysis ateach time point from post operative day (POD) 1-7. In this studyisoflurane anesthesia was employed. Vehicle treated 15-PGDH knockout(KO) mice were also used as an additional comparator.

FIG. 62 shows a graph depicting the pre-operative body weights of theFVB mice used for analysis of liver regeneration on post-operative days2, 3, 4 and 7. SW033291 and control treated mice used on each day arewell matched.

FIG. 63 shows a graph depicting the weight of the resected liver segment(resected LWt) from mice treated with either SW033291 or vehicle controland assayed for liver regeneration on post-operative days (POD) 2, 3, 4,and 7. Weight of the resected livers is well matched between control anddrug treated mice on each day, except on day 7, when the weight of theresected liver was greater in the SW033291 treated than in the controlmice.

FIG. 64 shows a graph depicting the liver weights attained(Regenerated_LWt) post partial hepatectomy in SW033291 and control miceon post-operative days 2, 3, 4 and 7 (POD 2, 3, 4, 7). SW033291 treatedmice show significantly greater liver weights versus control mice at alltime points, with SW033291 treated mice having approximately 25% greaterliver weight on post-operative day 7 than control mice.

FIG. 65 shows a graph depicting the liver to body weight ratios attained(LBWR) post partial hepatectomy in SW033291 and control mice onpost-operative days 2, 3, 4 and 7 (POD 2, 3, 4, 7). SW033291 treatedmice show significantly greater liver to body weight ratios versuscontrol mice at all time points, with SW033291 treated mice havingapproximately 20% greater liver to body weight ratio on post-operativeday 7 than control mice.

FIG. 66 shows “box and whisker” plot comparing liver to body weightratio's on post-operative day 4 following partial hepatectomy of FVBmice treated twice daily with SWO33291 5 mg/kg or with vehicle control,with 10 mice in each arm. Thick bars denote population median. Upper boxmargin denotes lower boundary of the highest quartile. Lower box margindenotes upper boundary of the lowest quartile. SW033291 treated miceshow a significantly increased liver to body weight ratio at P=0.004.

FIG. 67 shows “box and whisker” plot comparing liver to body weightratio's on post-operative day 7 following partial hepatectomy of FVBmice treated twice daily with SWO33291 5 mg/kg or with vehicle control,with 10 mice in each arm. Thick bars denote population median. Upper boxmargin denotes lower boundary of the highest quartile. Lower box margindenotes upper boundary of the lowest quartile. SW033291 treated miceshow a significantly increased liver to body weight ratio at P=0.001.

FIG. 68 shows “box and whisker” plot comparing liver to body weightratio's on post-operative day 4 following partial hepatectomy of FVBmice treated twice daily with SW033291 5 mg/kg or with vehicle control,with 10 mice in each arm. Also shown is the liver to body weight ratioon post-operative day 4 of 15-PGDH knockout mice (PGDH-KO) treated withvehicle only. Thick bars denote population median. Upper box margindenotes lower boundary of the highest quartile. Lower box margin denotesupper boundary of the lowest quartile. SW033291 treated mice show asignificantly increased liver to body weight ratio at P=0.001. 15-PGDHknockout mice also show greater a greater liver to body weight ratiothan do vehicle treated 15-PGDH wild-type mice, supporting that theliver regeneration activity of SW033291 is mediated through inhibitionof 15-PGDH. The larger effect of 15-PGDH gene knockout suggests furtherincrease in effect of SW033291 may be attainable with additionalmodification of dosing schedule and delivery.

FIG. 69 shows visualization of S-phase cells following partialhepatectomy on post-operative day 2 in livers of SW033291 treated andvehicle treated control mice. Mice were injected with BrdU at 50 mg/kgIP 2 hours before sacrifice, and then S-phase cells were visualized bystaining the livers with an antibody that detects BrdU that has beenincorporated into DNA. Representative fields at 10× magnification showthe clear increase in numbers of BrdU positive cells in the SW033291treated liver.

FIG. 70 shows high powered (40X) views of representative fields from thestudy of FIG. 69 .

FIG. 71 shows “box and whiskers” plot comparing percent of BrdU positivecells in livers of SW033291 treated versus vehicle control treated miceon post-operative day 2 following partial hepatectomy. Plotted in eachgroup are the percent BrdU positive cells from 100 random high poweredfields (40× magnification) counted as 10 fields from each of 10 drugtreated and each of 10 control vehicle treated mice. Heavy black barsshow median values of each distribution. Upper box margin denotes lowerboundary of the highest quartile. Lower box margin denotes upperboundary of the lowest quartile. SW033291 show a greater than 2-foldincrease in median S-phase cells on post-operative day 2 (P<0.05).

Example 9 Analysis of Effect of SW033291 on Survival FollowingAcetaminophen (Tylenol) Overdose

This Example provides data showing effects of SW033291 in mediatingresistance to lethal doses of the liver toxin acetaminophen (Tylenol).

In the study, 11 week old female C57BL/6J mice are injected IP with asuspension of acetaminophen in phosphate buffered saline administered atthe LD50 dose of 600 mg/kg.

Table 10 provides a tabular summary of the number of mice surviving outof an initial cohort of 6 mice that are all treated with acetaminophen(Tylenol) in phosphate buffered saline administered IP at the LD50 doseof 600 mg/kg.

TABLE 10 Treatment 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 1 does/daily, First dose immediately after Tylenolinjection Saline 6 6 5 4 3 3 3 3 3 3 3 5 mg/kg 6 6 6 4 4 4 4 4 4 4 4SW033291 2 dose/daily, First dose immediately after Tylenol injectionSaline 6 6 6 4 3 3 3 3 3 3 3 5 mg/kg 6 6 6 4 3 3 3 3 3 3 3 5W033291

Test mice are additionally treated with SW033291 5 mg/kg IP in a vehicleof 10% Ethanol, 5% Cremophor EL, 85% D5W beginning immediately followingacetaminophen and continued once daily, or twice daily. Control mice areadditionally treated with vehicle alone once daily or twice daily.Survival is recorded from the 0 time point of administration ofacetaminophen through 120 hours following. No difference is notedbetween survival of SW033291 treated and control mice.

Table 11 shows a summary of the number of mice surviving out of aninitial cohort of 12 eleven week old C57BL/6J female mice that are alltreated with acetaminophen (Tylenol) in phosphate buffered salineadministered IP at the LD50 dose of 600 mg/kg, Mice are additionallytreated with either SW033291 or vehicle control.

TABLE 11 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 5 mg/kg 12 12 12 11 10 10 10 10 10 10 10 SW033291 1212 12 6 5 5 5 5 5 5 5 Saline

SW033291 5 mg/kg was administered IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W twice daily (bid) beginning 48 hours prior toacetaminophen injection and continuing for 48 hours followingacetaminophen injection for 9 doses total. At 120 hours postacetaminophen injection, 10 of 12 mice have survived in the SW033291treated cohort versus 5 of 12 mice in the vehicle control treatedcohort, P=0.045 in a one-tailed Fisher's exact test. Thuspre-administration of SW033291 protects from the lethal hepatotoxicityof acetaminophen.

Table 12 shows a summary of the number of mice surviving out of aninitial cohort of 6 eleven week old C57BL/6J female mice that aretreated with acetaminophen (Tylenol) in phosphate buffered salineadministered IP at the LD50 dose of 600 mg/kg.

TABLE 12 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 5 mg/kg 6 6 5 4 3 3 3 3 3 3 3 SW033291 6 6 5 3 2 2 22 2 2 2 Saline

Mice are additionally treated with either SW033291 or vehicle control.SW033291 5 mg/kg was administered IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W twice daily (bid) beginning 3 hours prior toacetaminophen injection and continuing at time 0 through 48 hoursfollowing acetaminophen injection for 6 total doses. At 120 hours postacetaminophen injection 3 of 6 mice have survived in the SW033291treated cohort versus 2 of 6 mice in the vehicle control treated cohort.

Table 13 shows a summary of the number of mice surviving out of aninitial cohort of 7 C57BL/6J 25 week old female 15-PGDH wild-type (WT)or 7 C57BL/6J 25 week old female 15-PGDH knockout (KO) mice treated withacetaminophen (Tylenol) in phosphate buffered saline administered IP atthe LD50 dose of 600 mg/kg.

TABLE 13 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr WT 7 7 7 5 4 3 3 3 3 3 3 KO 7 7 7 6 6 6 6 6 6 6 6

At 120 hours post acetaminophen injection, 6 of 7 knockout mice surviveversus 3 of 7 wild-type mice. Increased survival of 15-PGDH knockoutmice is consistent with the survival benefit of SW033291 being mediatedthrough inhibition of 15-PGDH.

Example 10 Analysis of Effect of SW033291 on Dextan Sodium Sulfate (DSS)Induced Colitis

This Example provides data from studies of the effect of SW033291 onprevention of induction of colitis in the dextran sodium sulfate (DSS)treated mouse. In the study, 8-12 week old FVB male mice are fed with 2%DSS in drinking water for days 1-7, and then switched to normal drinkingwater beginning on day 8, and continued through day 22. Mice are treatedwith twice daily SW033291 5 mg/kg IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W, at 125 ug/200 ul, versus with vehicle alone.Clinical scoring (body weight, rectal bleeding, stool consistency) isrecorded daily, endoscopic scoring (ulcer number, mucosal thickening,and vascular pattern) is assessed on days 8, 11, 15. Mice are sacrificedon days 1, 8, 15 and 22 for assessment of colon length, colon weight,ulcer number, ulcer area, and crypt damage.

Table 14 shows summary of the baseline properties of age and weight ofthe 24 SW033291 treated mice and the 24 control group mice used in thestudy. Also provided is baseline characteristics of 4 FVB male 15-PGDHknockout (KO) mice that are used as a comparator group.

TABLE 14 FVB PGDH WT/ KO male mice 8-12 weeks old DSS Study WT-ControlWT-Treatment KO p-value Number 24 24 4 Sex M M M Age (Days) 74.1 ± 3.7 74.2 ± 4.0  73.9 ± 3.4 0.655 Weight (gm) 26.3 ± 1.19 26.8 ± 1.78 27.4 ±1.4 0.391

FIG. 72 shows a graph of the average changes from baseline weight of thecohort of control versus SW033291 treated mice across the 22 days of thestudy. SW033291 treated mice show greater weight at all time points, andin particular, show faster weight gain after washout of DSS then do thecontrol mice, P=0.001.

FIG. 73 shows a graph of the daily Disease Activity Index (DAI) of thecohort of control versus SW033291 treated mice across the 22 days of thestudy. The Disease Activity Index is calculated as an equally weightedaverage of the change from baseline weight, the consistency of stool,and the presence of rectal bleeding, with each component normalized tospan an identical numerical range. SW033291 treated mice show a lowerDisease Activity Index than do control on each day of the study,P<0.001.

FIG. 74 shows the design of the study in which colonoscopic examinationof the left colon, up to the splenic flexure, was performed on live miceon days 8, 11 and 15, under isoflurane anesthesia. In addition,post-mortem colonoscopy of the full colon was performed on two SW033291treated and two control treated mice on day 15, with findings confirmingthat DSS induced ulcerations are largely confined to the descendingcolon distal to the splenic flexure.

FIGS. 75 (A-B) show at bottom left the colon as visualized duringcolonoscopy of a DSS treated control mouse that shows loss of themucosal vascular pattern and a gross ulceration. At bottom right isshown the colonoscopic findings of a DSS treated mouse receivingSW033291, with only a small ulcer and with maintenance of the normalmucosal vascular pattern otherwise. Graph at top shows numbers of ulcerspresent on days 8, 11, and 15 in the control versus SW033291 treatedmice. SW033291 treatment prevents two-thirds of ulcer formation.Additional studies of 15-PGDH knockout mice show that 15-PGDH geneknockout prevents 95% of colon ulcer formation. These findings supportthat the colitis prevention activity of SW033291 is mediated through itsactivity as a 15-PGDH inhibitor, and suggest further modifications ofdrug dosing and delivery may provide added colitis prevention.

FIG. 76 shows quantitation of ulcer burden on day 15 of DSS treated miceas determined by embedding the full length of the formalin fixed colonsof mice in paraffin blocks, and then microscopic inspection of a random5 μm section along the full colon length for visualization andmeasurement of ulcerated mucosa. The graph shows that the average lengthof ulcerated mucosa is 4.48 mm per colon section in control mice (N=9mice) and is reduced by 61% to a length of 1.74 mm per colon section inSW033291 (drug) treated mice (N=6 mice), P=0.045. Again, 15-PGDH geneknockout (KO) is highly effective in preventing colon ulceration,supporting that the therapeutic effect of SW033291 is mediated throughinhibition of 15-PGDH.

FIGS. 77 (A-B) shows examples of scoring murine colonic mucosa accordingto the Murine Endoscopic Index of Colitis Severity (MEICS) (Becker C. etal. Gut 2005; 54: 950-954). At top (A) is shown the colonoscopicfindings and MEICS scoring for a DSS treated mouse receiving SW033291.At bottom (B) is shown the colonoscopic findings and MEICS scoring of aDSS treated mouse receiving vehicle only.

FIG. 78 shows graphs of the MEICS scores for DSS treated mice receivingSW033291 (treatment) versus vehicle (control). MEICS scores showsignificantly less colitis activity in SW033291 treated mice on days 8,11 and 15 of the study.

In addition to the gross visual inspection and scoring of colitisactivity by the MEICS index, full length colons of mice were formalinfixed and paraffin embedded, and microscopic scoring of crypt damage wasperformed using the 0-4 severity scale of Cooper H S. Et al., LabInvest. 1993; 69:238-249. For this analysis, the colons were dividedinto 3 segments of proximal, middle, and distal colon, eachapproximately 1.6 cm in length, with each segment was further subdividedinto 4 sections each approximately 4 mm in length. For each section thecrypt damage severity score was multiplied by the length in mm of thedamaged area, creating a 0-16 cryptitis severity index. An averagecryptitis severity index was calculated for each segment (proximal,middle, and distal colon), and the summed whole colon cryptitis severityindex was determined on a scale of 0-48 for each mouse colon. Inparallel with the visual MEICS score, the microscopic cryptitis severityindex on day 8 of the DSS protocol was significantly greater in controlmice (value of 9.49) than in the SW033291 treated mice (value of 3.16),P<0.05 (data described but not shown in the figure)..

FIG. 79 shows assessment of the effect of SW033291 on maintaining DNAsynthesis in the colonic mucosa of DSS treated mice. Mice were injectedwith BrdU at 100 mg/kg IP 3 hours before sacrifice and then full lengthcolons were formalin fixed and embedded in paraffin. S-phase cells, thathave incorporated BrdU into DNA, were visualized by immuno-fluorescentstaining of Sum thick sections with an antibody that detects the BrdU.Colonic crypts were visualized by immuno-fluorescent staining with anantibody to the epithelial marker E-Cadherin. Photographic insets showphotomicrographs of high powered fields taken from the mid-colon on day8 of the DSS protocol from control mice, SW033291 treated mice(treatment) and 15-PGDH knockout mice (KO). Red immune-fluorescenceidentifies BrdU positive nuclei, and green immune-fluorescenceidentifies E-Cadherin positive colonocytes. The number of BrdU positivecell per crypt is determined by counting the number of dual labeled redand green cells per average crypt. Green only cells that are not inS-phase are not counted, and red only cells, that are likely stromalcells outside of crypts, are also not counted. On the photomicrographshown crypts are displayed as vertically oriented in control andSW033291 treated mice, and crypts are displayed as horizontally orientedin the 15-PGDH knockout mice. In the photographs the numbers of S-phasecells are fewest in the control mice and are increased in the SW033291treated mice, and increased further in the knockout mice. In theparticular photographs shown, the crypts from control mice both lackS-phase cells and are also visually decreased in height; whereas, cryptheight is increased in the crypts shown from SW033291 treated mice, andcrypt heights is increased further in the crypts shown from 15-PGDHknockout mice. The graph depicts the sum of the average number of BrdUpositive cells per crypt in the distal colon plus the average number ofBrdU positive cells per crypt middle colons of control (Cn), SW033219treated (Tx), and 15-PGDH knockout mice (KO) on day 1, day 8, and day 15of the DSS treatment protocol. On day 8, SW033291 treated micedemonstrate 5.7-fold greater numbers of BrdU positive cells than docontrol mice, which have lost 85% of the day 1 value of BrdU positivecells per crypt. 15-PGDH knockout mice show no loss of BrdU positivecells in the crypt on day 8, consistent with the protective effect ofSW033291 being mediated by inhibition of 15-PGDH.

Table 15 shows a summary of colon length (in cm) in DSS treated micesacrificed on days 8, 15 and 22, in SW033291 treated mice, versusvehicle treated control mice, versus 15-PGDH knockout (KO) mice, whereshortening of the colon is a measure of disease activity.

TABLE 15 Colon length shortening may be correlated to severity of thecolon ulceration Time Point Control SW033291 KO P-value Baseline 8.3 +0.2 8.4 + 0.2 0.71 Day 8 6.6 + 0.4 6.6 + 0.1 1.0 Day 15 7.1 + 0.1 7.5 +0.1 8.5 + 0.1 0.001 Day 22 7.4 + 0.2 8.6 + 0.3 0.012

Vehicle treated control mice show significantly greater colon shorteningat day 22 versus SW033291 treated mice, P=0.012. This comparison is alsoshown graphically in FIG. 80 .

Table 16 shows a summary on day of sacrifice of mouse weights (gms) andcolon lengths (cm) for DSS treated mice receiving SW033291 or vehiclecontrol.

TABLE 16 Vehicle SW033291 KO Wt @ sacrifice-gm Time Point Baseline26.3 + 0.7 25.9 + 0.7 29.2 + 1.3 Day 8 25.4 + 0.7 26.4 + 0.5 Day 1524.4 + 0.5 25.2 + 0.9 Day 22 * 26.3 + 0.7 28.2 + 0.5 Colon length-cmTime Point Baseline  8.3 + 0.2  8.4 + 0.2  8.5 + 0.1 Day 8  6.6 + 0.4 6.6 + 0.1 Day 15  7.1 + 0.1  7.5 + 0.1 Day 22 *  7.4 + 0.2  8.6 + 0.3

On day 22 SW033291 treated mice show greater body weight and greatercolon lengths, indicative of therapeutic effect of SW033291 inprotecting against DSS induced colitis.

Example 11: Analysis of Analogues of Lead Compound SW054384, a 15-PGDHActivator

This Example provides data on a group of structural analogues ofSW054384. Data on Table 17 characterize analogues obtained by CaseWestern Reserve University from a chemical library shared with theUniversity of Cincinnati. Data on Table 18 are analogues ordered fromcommercial sources. Data on Table 19 are analogues held in chemicallibraries or synthesized by members of the inventors group at Universityof Texas Southwestern.

TABLE 17 V9M V9M LS174T 1S174T V503 V503 reporter reporter reporterreporter reporter reporter activity activity activity activity activityactivity Structures Name (2.5 μM) (7.5 μM) (2.5 μM) (7.5 μM) (2.5 μM)(7.5 μM)

292301 12.92 23.57 12.07 −3.67 −0.91 −3.60

396087 −8.51 10.08 4.37 10.18 17.23 30.79

407572 −8.45 −6.12 2.22 6.94 17.22 35.88

408961 −12.39 0.56 2.46 −4.51 11.71 17.61

414177 10.61 7.72 9.51 −0.62 −2.80 −5.73

414183 −9.15 −3.89 5.35 −4.77 11.49 37.17

414195 −13.95 −16.76 −1.15 2.34 13.14 19.47

474738 −7.27 3.48 −0.36 3.50 17.03 24.59

474759 2.58 6.40 5.54 −0.03 −5.04 −12.72

503270 −4.47 24.06 12.29 32.82 29.88 42.14

537833 −7.54 −7.44 4.94 9.04 42.31 73.22

539430 21.27 21.35 5.62 10.45 38.75 103.04

543512 36.82 79.64 18.05 8.33 −4.56 −4.26

543716 4.20 −0.14 3.26 −2.08 −8.04 −8.60

742790 13.79 23.09 12.77 17.32 5.94 34.38

771064 43.32 −19.19 −2.11 −29.63 −13.75 −24.80

771069 −22.56 −54.66 −29.88 −82.88 −62.71 −90.88

771074 13.50 24.69 20.67 20.60 22.96 39.52

771081 −14.22 7.58 5.74 15.38 31.87 51.68

771130 8.67 5.84 8.66 −0.48 −10.19 −3.48

771132 −2.26 12.24 13.98 13.54 24.75 40.76

771138 −4.36 6.75 13.64 9.23 34.44 72.58

771146 −38.07 −78.30 −31.58 −68.78 −49.73 −86.60

771432 −7.11 −3.13 3.86 4.24 33.72 42.82

785160 −15.99 21.00 5.93 48.64 33.85 44.75

785161 0.54 3.48 10.68 −41.92 56.72 −52.51

785254 −16.05 −8.48 2.93 4.29 16.14 28.28

918280 −16.26 −77.26 −21.21 −72.09 −35.68 −74.96

919570 87.71 127.26 78.80 102.68 80.94 104.40

919576 49.89 63.03 17.49 30.74 49.20 73.74

920847 9.26 17.73 6.18 2.97 −4.47 −10.57

924740 −0.54 6.33 7.69 16.73 20.16 52.17

924787 −1.45 18.08 4.32 13.10 21.87 47.97

928861 64.65 134.49 74.89 124.37 73.17 138.59

939417 −11.15 −9.81 6.50 −1.64 12.58 16.25

967134 −10.12 −2.64 5.11 5.73 18.43 36.12

1002065 7.38 18.64 9.90 15.22 38.12 52.53

1002654 −2.58 −3.96 5.51 −1.24 0.78 19.23

1003012 −10.45 −0.90 5.54 1977. 34.43 58.54

1003132 −5.60 −0.21 5.16 1.73 15.40 16.05

1003378 −4.42 −3.27 7.30 1.27 7.23 16.73

1003429 12.60 4.17 8.82 −6.88 −7.46 −19.23

1003669 6.30 7.58 5.98 −1.93 1.22 27.08

1004292 0.88 −0.21 20.34 0.14 23.50 21.16

1004571 −6.09 10.99 12.35 29.28 29.09 85.80

1004631 −3.66 12.03 4.51 45.32 52.12 87.08

TABLE 18 V9M-SC3 V9M-SC3 Ls174T-SC1 Ls174T-SC1 V503-3H9-7 V503-3H9-7Enzyme Enzyme Reporter Reporter reporter reporter reporter reporterinhibition inhibition activity activity % activity, % activity, %activity, % activity, % % % Structure CAS # (2.5 μM) (%) (7.5 μM) (2.5μM) (7.5 μM) (2.5 μM) (7.5 μM) (2.5 μM) (7.5 μM)

333447-10-2 −0.5 10 7.54 14.44 76.87 158.91 27.01 46.46

332419-63-3 2.5 2 12.44 9.81 1.65 46.28 36.59 47.22

311333-86-5 53 117.25 29.97 53.41 102.18 131.25 33.99 34.01

428460-22-4 1.5 15.25 −7.63 −1.99 1.39 47.04 15.05 19.30

426231-26-7 10 2.5 16.17 22.07 64.02 113.49 39.81 56.40

339589-55-8 −7 8 −5.54 8.81 63.97 52.49 19.14 21.99

339589-53-6 −1 4.25 51.18 89.74 199.56 105.28 35.46 37.35

530107-57-4 59.5 116.5 5.45 8.27 52.62 84.39 21.74 9.25

333449-58-4 6.5 71.25 −5.90 −3.99 50.08 20.72 9.72 −3.12

333446-93-8 16.5 44 12.90 13.53 53.78 44.10 −5.31 5.85

333450-97-8

311315-25-0 62.59 75.79 18.69 34.70

701217-00-7 −2.05 79.49 −06.89 −0.54 20.078 33.42 10.07 13.12

592469-85-7 −9.74 18.46 3.77 13.99 10.16 5.30 6.23 17.24

526206-05-3 23.08 113.85 68.46 145.94 110.21 197.50 41.97 70.19

428473-22-7 −17.95 2.051 −15.50 10.58 19.09 19.81 3.76 8.67

418783-65-0 0.51 67.18 −12.59 5.81 11.63 6.68 10.85 12.12

367929-80-4 −27.79 −19 18.80 −17.98 −0.87 −6.22 8.95 34.80

335392-39-7 −12.82 7.18 −0.54 14.32 18.89 21.95 11.00 5.37

331727-07-2 28.21 120 −7.53 9.47 8.13 1.14 9.65 17.53

328012-52-8 −15.38 0.51 127.66 175.99 242.64 −4.89 67.39 82.06

310875-38-8 −8.72 −16.41 27.66 101.94 64.11 147.62 23.51 33.40

833428-03-8 −4.10 17.44 25.08 47.36 96.38 149.97 16.39 49.25

528580-69-0 4.71 −0.5 11.49 −4.81 66.03 110.41 −8.22 10.23

461439-84-9 −30.29 −41.75 4.83 −34.97 1.57 5.41 9.28 3.35

680599-58-0 −5.29 −22.5 11.489 −13.81 16.66 28.86 31.81 50.71

505072-23-1 94.71 71.5 100.91 109.26 242.42 238.88 67.44 84.57

527699-66-7 −31.03 −37.25 7.31 −29.61 38.78 70.92 −20.58 27.93

TABLE 19 V9M- V9M- Ls174T- Ls174T- V503- V503- SC3 SC3 SC1 SC1 3H9-73H9-7 Decrease Reporter Reporter reporter reporter reporter reporterEnzyme PGE-2 Decrease Decrease activity activity activity, activity,activity, activity, inhibition Enzyme (IL- Cell of (2.5 (7.5 % % % % %inhibition Enzyme 1beta- viability coloning μM) μM), (2.5 (7.5 (2.5 (7.5(2.5 % (7.5 inhibition A549, in A549 formation Structure UTSW ID (%) %μM) μM) μM) μM) μM) μM) (IC50) %) cell (%) %

SW054384 116.34 147.89 129.91 155.75 148.89 157.49 20.17 58.20 41.96

SW202939 75.34 127.84 75.46 166.46 26.60 27.30 53.54 78.32

SW202942 52.69 104.86 53.59 122.86 37.26 86.87 48.39 64.86

SW202945 28.49 89.98 45.67 134.54 33.99 127.43 8.83 11.07

SW202949 5.76 −5.69 −1.58 −5.87 −12.46 5.32 12.52 34.37

SW202950 12.59 23.87 −2.70 10.69 −6.56 12.95 46.26 56.39

SW202953 12.97 34.79 20.69 30.69 31.98 50.93 8.81 15.06

SW202954 2.70 4.80 −4.59 −6.99 −8.94 2.39 6.02 9.88

SW202944 −0.95 −3.73 32.47 15.76 130.94 94.57 28.28 57.78

SW202948 −21.99 −23.05 −6.73 8.36 16.44 7.54 28.27 56.73

SW202947 21.89 2.57 52.05 21.22 142.96 97.85 28.80 59.19

SW202952 −20.74 −19.53 −10.30 10.58 26.18 7.16 29.01 57.03

SW202940 146.58 181.17 147.86 210.71 180.73 182.58 24.30 63.29 ~5 μM80.46 45.18

SW202942 1.00 32.05 46.96 86.98 95.39 129.34 −3.72 28.87

SW202943 7.12 17.84 33.07 55.73 72.27 146.87 −7.42 21.84

SW202946 64.59 −41.52 105.33 −23.62 136.73 34.27 81.60 89.84

SW202951 4.56 17.661 34.26 47.64 97.53 131.49 −15.43 −8.59 −1.01

SW122063 1.67 4.80 22.56 19.86 42.51 44.12 −12.23 −10.49

SW202938 154.92 230.12 216.99 240.95 309.09 300.99 52.66 75.64 2.67 μM−47.55

SW202941 −10.89 2.57 −23.79 −43.20 75.94 83.54 10.86 35.61

SW202965 −30.92 −25.80 −23.51 3.52 23.76 98.06 11.26 33.13

SW202966 −0.84 67.67 1.82 78.15 115.05 248.97 44.66 61.39

SW202967 −15.98 0 −11.69 9.37 34.05 69.62 −11.56 0.94

SW202968 −21.10 −7.36 −17.50 6.08 35.18 53.87 −7.09 0.78

SW202969 −20.96 −14.30 −15.87 17.90 62.42 126.16 0.91 9.43

SW202970 32.74 17.81 47.94 55.89 179.12 121.93 17.46 48.04

SW202971 −14.51 9.67 −7.91 34.55 85.50 169.04 −0.72 −13.18

SW202972 −15.42 0.14 −17.45 16.64 39.42 100.21 −6.29 −2.94

SW202973 −15.63 5.61 −16.38 19.29 41.53 106.91 3.3 13.55

SW202974 −9.04 35.90 −8.77 34.79 59.88 179.28 −4.42 24.85

SW202977 −8.69 26.22 −16.61 15.09 16.92 85.24 −14.95 −8.63

SW202978 −13.81 −0.07 −10.05 20.43 46.77 116.46 4.14 3.12

SW202979 −14.44 10.79 −8.92 17.83 41.64 133.05 3.92 6.24

SW202980 −27.55 −13.39 −7.34 5.17 11.41 50.62 −4.19 1.44

SW202985 −27.34 −21.87 −4.89 26.32 79.56 149.89 13.79 30.87

SW202986 −22.86 −9.60 0.21 33.80 60.17 175.41 −2.22 2.58

SW202987 −22.01 −21.03 −8.30 20.65 70.129 145.13 4.61 6.13

SW202988 7.36 54.41 40.50 125.44 125.39 258.27 10.27 33.70 ~10 μM −16.79

SW202989 187.65 183.80 158.81 171.45 207.62 213.94 81.32 89.86 427 nM69.40 30.59

SW202990 3.57 23.49 32.01 106.56 172.00 214.22 22.88 47.16

SW202975 −26.64 −8.97 −7.32 17.21 47.35 116.21 −14.82 4.53

SW202976 −18.51 9.60 −9.97 30.14 36.75 138.31 7.87 29.78

SW202991 −25.03 −7.50 −15.58 2.09 −6.08 8.90 −14.71 −6

SW203680 13.98 25.21 11.73 45.97 10.43 32.98 34.82 62.10

SW203681 17.99 121.71 120.56 151.88 90.42 72.37 36.77 64.87

SW131633 77.21 140.98 8.42 149.84 103.52 −40.56 23.13 35.21

SW203682 15.31 22.45 187.26 13.76 −32.19 79.19 15.73 36.23

SW203683 137.32 190.83 3.76 209.77 103.81 −41.03 23.41 57.09 ~7 μM −3.41

SW131635 11.39 15.05 110.93 14.07 −37.66 14.39 11.80 35.44

SW203684 50.72 74.89 170.37 119.07 81.19 52.46 36.35 64.72

SW203685 99.95 204.48 22.34 253.97 121.53 −33.18 44.34 73.30 5 μM 17.84

SW203686 15.05 30.30 116.07 28.49 −11.03 65.60 14.86 34.24

SW202942 85.24 132.15 30.25 143.05 69.32 3.29 36.78 53.24

SW203687 28.25 47.51 1.75 56.00 45.74 −41.65 15.07 33.55

SW203688 12.10 6.04 12.59 14.75 −28.10 −99.52 13.13 39.96

SW203691 7.78 19.82 5.47 22.03 47.07 90.04 2.62 3.12 36.76 0.00

SW203703 108.15 105.91 85.80 80.20 142.81 77.97 26.50 58.88 31.90 −1.97

SW203704 8.71 8.04 8.64 12.47 52.84 37.84 4.19 7.26 22.33 −2.70

SW125991 77.40 74.63 67.28 83.33 160.61 148.71 11.61 31.62 31.33 −2.13

SW203736 132.36 145.08 126.75 155.67 244.43 203.63 1.70 24.86 31.86−3.06 2.5 μM (3.12%), 7.5 μM (5.18%)

SW203737 93.37 103.47 76.65 85.09 175.94 153.58 23.81 42.45 47.78 −2.562.5 μM (10.25%), 7.5 μM (82.15%)

SW208001 19.59 23.31 −0.61 1.87 −4.07 6.68 21.79 27.74

SW208002 −12.39 −21.84 −14.51 −14.57 −15.65 −16.54 34.73 59.23

SW208003 13.32 −11.46 37.00 45.62 117.02 75.883 15.19 20.91

SW208004 43.68 76.14 62.19 118.21 146.61 176.82 52.33 57.00

SW208005 42.44 19.75 63.86 53.93 102.72 61.24 44.10 52.02

SW208006 34.85 0.15 38.20 42.18 89.69 43.90 15.71 24.78

SW208007 −25.48 −26.41 −9.01 −5.15 −3.17 −2.10 48.27 38.41

SW208000 33.54 36.63 41.98 70.53 121.50 138.25 5.82 12.47

SW208008 −5.96 −0.92 −2.16 10.01 31.93 39.63 58.70 70.90

SW207997 59.48 71.03 87.60 94.08 148.77 156.90 −7.08 −0.86

SW207998 53.36 58.94 65.15 76.99 143.67 147.18 8.09 14.38

SW207999 128.11 124.32 98.00 122.98 123.99 110.28 −0.86 3.34

Data provided include level of induction of a 15-PGDH-luciferase fusiongene reporter, recorded as % induction of luciferase activity over basallevel, in three colon cancer cell lines, V9m, V503, and LS174T,engineered to contain the reporter, and treated with either 2.5 uM or7.5 uM compound. Also recorded for some compounds is the inhibition ofenzyme activity of recombinant 15-PGDH protein treated with 2.5 μM or7.5 μM compound. Also recorded for some compounds is the IC₅₀ of eachcompound for inhibiting enzymatic activity of recombinant 15-PGDH in anin vitro assay. Additionally, for selected compounds is recorded thedecrease in PGE₂ levels in the media of compound treated A549 cells thathave been stimulated with IL-1 beta. Additionally recorded for selectedcompounds is the effect on A549 cell viability as measured by aCellTiter-Glo assay, and the effect of compounds on A549 cell colonyformation.

We first note that the amino group participating in the peptide bond inSW054384 can be modified as shown in compound MCD-03-025, and that thederived compound retains the ability to activate expression of the15-PGDH-luciferease reporter in reporter cell lines, and indeed showslesser inhibition of recombinant 15-PGDH at 2.μM in the test tube thandoes parent compound SW054384.

We also note that addition to the phenyl ring of SW054384 of fluorine(SW203736) or bromine (SW203737), is well tolerated, and yields acompound that is active in inducing 15-PGDH-luciferase reporter activityin cell lines, is similar or improved compared to parental SW054383 inminimally inhibiting recombinant 15-PGDH at 2.5 uM, and is similar orimproved versus parental SW054384 in decreasing PGE₂ levels in the mediaof compound treated A549 cells that have been stimulated with IL-1 beta.The phenyl ring of SW054384 also tolerates addition of a methoxy group(SW202940), which yields a compound that is that is active in inducing15-PGDH-luciferase reporter activity in cell lines, and is similar toparental SW054384 in not inhibiting recombinant 15-PGDH at 2.5 uM.

We also note the favorable properties of compound SW125591, also denotedSW125991, which converts the nitrogen in the SW054384 peptide bond froman (Aryl)-NH— group into a cyclic amine. This compound retains activityin induction of the 15-PGDH-luciferase reporter assay. It shows lessinhibition of 15-PGDH at high compound concentration than does the leadenzyme activator SW054384. SW125991 shows similar activity to SW054384in reducing PGE₂ levels in IL1-beta stimulated A549 cells. SW125991shows no toxicity as assessed by effect on CellTiter-Glo assays done at24 hours. Moreover, SW125991 shows marked improvement in metabolicstability.

We also note the favorable properties of compounds SW207997, SW207998,and SW207998. These three compounds, like SW125991, have all convertedthe nitrogen in the SW054384 peptide bond from an (Aryl)-NH— group intoa cyclic amine. In addition, SW207997, SW207998, and SW207998, have alladded a methoxy group to the phenyl ring in SW054384. SW207997,SW207998, and SW207998 all show activity equal to, or in some assaysgreater than, SW054384 in inducing the 15-PGDH-luciferase gene fusionreporter construct, and all show much less inhibition of enzymaticactivity of 15-PGDH at 2.5 μM and 7.5 μM than does SW054384.

Example 12

The following Example describes the synthesis of SW054384 and analoguesthereof as well as provides mass spectrometry NMR confirmation of thestructures.

General Procedure 1:

Procedure

(Hetero)aryl amine was dissolved in 1:1 mixture of DMF/dioxane (1.215 Mbased on amine) and cooled to 0° C. Bromoacetyl bromide (1.26 equiv)added drop-wise and allowed to warm to room temperature overnight.Dioxane and excess acid bromide were removed under reduced pressure, and2,5-dimethoxy aniline (3.5 equiv) was added. The crude material washeated to 120° C. for three hours. The reaction was cooled to roomtemperature, diluted with EtOAc, and filtered through celite. Thefiltrate was washed with water (3×), sodium bicarbonate, and brine. Theorganic phase was dried with MgSO4 and concentrated to give crude amide(1). The product was further purified by flash chromatography to affordpure N-aryl glycinanide 1.

N-Aryl glycinamide (1) was dissolved in CH₂Cl₂ (1M). Pyridine (4.9equiv) and sulfonyl chloride (2) were added (1.3 equiv) and the reactionwas stirred overnight. The solution was diluted with EtOAc, washed withwater (3×) and brine, dried over MgSO4 and concentrated under reducedpressure to give crude sulfonamide 3. The product was purified by flashchromatography.

General Procedure 2:

Procedure

Aniline 4 (1 equiv) was dissolved in CH₂Cl₂ (1 M based on 4). DMAP (0.3equiv) and pyridine (4.9 equiv) were added followed by sulfonyl chloride2 (1 equiv). The reaction was stirred for 36 hours, diluted with CH₂Cl₂and washed with water, HCl (1 M), sodium bicarbonate, and brine. Theorganic layer was dried over MgSO4 and solvent was removed under reducedpressure to afford 5.

Without purification, secondary sulfonamide 5 (1 equiv) was dissolved ina solution of DMF (0.1 M) and ethyl bromo acetate (4 equiv). Thissolution was added drop-wise to sodium hydride at 0° C. After bubblingceased, the reaction was warmed to room temperature and left stirringovernight. After 12-18 hours, water was added and the aqueous phase wasextracted 3 times with CH₂Cl₂. The combined organic extracts were washedagain with water followed by brine, dried with MgSO4, and concentratedunder reduced pressure to provide an oil. Addition of hexanes resultedin precipitation of the crude product, which was isolated by filtrationand used without purification.

Ester 6 was dissolved in 3:3:1 ratio of MeOH:THF:Water. Lithiumhydroxide was added and the reaction was stirred until complete (1-3hrs). The reaction was concentrated under reduced pressure and thendiluted with CH₂Cl₂. The acid (7) was extracted into saturated sodiumbicarbonate. The aqueous layer was neutralized with 1M HCl (pH˜5) andextracted with CH₂Cl₂ to afford 7, which was used without purification.

General Procedure 2A

Acid 7 (1 equiv) was dissolved in EtOAc (2 vol). Pyridine (1 vol) andsecondary amine (1.1 equiv) were added followed by T3P (2 equiv, 50% inEtOAc) and reaction was stirred overnight. The reaction was quenchedwith HCl (0.5 M, 3 vol), and the mixture was diluted with EtOAc, washedwith water, bicarbonate, and brine, dried with sodium sulfate andconcentrated. Compound 8A was purified by flash chromatography.

General Procedure 2B

Primary amine (1 equiv), DMAP (0.3 equiv) and EDCI (1.3 equiv) wereadded to a solution of acid 7 (1 equiv) in CH₂Cl₂ (0.5 M). The reactionvial was purged with nitrogen stirred for 12-24 h. The reaction mixturewas diluted with CH₂Cl₂ and washed with brine, water, HCl (1 M), sodiumbicarbonate and brine again. The organic layer was dried with sodiumsulfate and concentrated to afford 8B. If needed, the compound waspurified by flash chromatography.

General Procedure 2C:

Alcohol (4 equiv), DMAP (0.3 equiv) and EDCI (1.1 equiv) were added to asolution of acid 7 (1 equiv) in CH₂Cl₂ (0.5 M) at 0° C. After fiveminutes the reaction was allowed to warm to room temperature and wasstirred until complete. The reaction mixture was diluted with CH₂Cl₂ andwashed with brine, HCl (1 M), and water. The organic phase was driedwith MgSO4 and concentrated to afford the pure ester 8C.

General Procedure 3

Procedure: Pyridine (1.1 equiv) was added to a solution of secondaryamine 9 in THF (0.5M), and reaction mixture was cooled to 0° C.Bromoacetyl bromide (1 equiv) was then added drop-wise and reaction waswarmed to room temperature. The reaction was stirred for two hours atthis temperature and then diluted with EtOAc and washed with water. Theorganic layer was dried with sodium sulfate and concentrated to affordbromo acetamide 10, which was purified by flash chromatography on silicagel.

General Procedure 3A: A solution of amide 10 in DMF (0.075 M) was addedto a solution of sulfonamide 12 (1.5 equiv; synthesis identical tosulfonamide 5 in Procedure 2), potassium carbonate (2 equiv), and DMF(0.075 M). The reaction was stirred overnight and then diluted withEtOAc and washed with water and brine. EtOAc was removed under reducedpressure to afford crude 12, which was purified by flash chromatographyon silica gel.

General Procedure 3B: A solution of amide 10 in DMF (0.075 M) was addedto a solution of aniline, potassium carbonate (2 equiv), and DMF (0.075M). The reaction was stirred overnight and was then diluted with EtOAcand washed with water and brine to afford amide 11 crude, which was usedwithout purification.

DMAP (0.3 equiv), pyridine (4.9 equiv) and then sulfonyl chloride (1equiv) were added to a solution of amide 11 in CH₂Cl₂ (1 M). Thereaction was stirred for 12-24 hours, diluted with CH₂Cl₂ and washedwith water, HCl (1 M), sodium bicarbonate, and brine. The organic layerwas dried over MgSO4 and compound concentrated under reduced pressure toafford sulfonamide 12, which was purified by flash chromatography.

Example 13: Properties of Selected Analogues of Lead Compound SW054384,a 15-PGDH Activator

This Example provides data on properties of selected analogues ofSW054384.

FIG. 81 shows structures of selected analogues of SW054384.

FIGS. 82 (A-C) show graphs that show the level of activity in inducingthe 15-PGDH-luciferase fusion reporter in three different test cell linebackgrounds, V9m, LS174T, and V503. Each compound was tested at twoconcentrations, 2.5 uM, and 7.5 uM. Y-axis is luciferase activity.Compounds active in inducing the 15-PGDH-luciferase reporter includeSW20370, SW203704, SW125991, SW203736, SW203737.

FIG. 83 shows activity of tested compounds in inhibiting enzymaticactivity of recombinant15-PGDH when tested at high concentrations of 2.5uM and 7.5 uM. SW125991 and SW203736 show lesser inhibitory activity athigh concentration than the lead 15-PGDH activator, SW054384. SW203737at these concentrations shows inhibitory activity against recombinant15-PGDH that is similar to SW054384.

FIGS. 84 (A-C) show at left (84A) activity in lowering PGE₂ levels inmedia of A549 cells that are stimulated to produce PGE₂ by treatmentwith IL1-beta. All of the compounds tested, including SW054384,SW203703, SW125991, SW203736, and SW203737, show activity in loweringPGE₂, consistent with their acting in vivo as inducers of 15-PGDHactivity. FIG. 84B shows at right that none of the tested compoundsdemonstrate any toxicity against A549 cells as assessed by CellTitre-Gloassay levels at 24 hours of treatment. FIG. 84C shows at bottomphotographs of A549 cells tested in the CellTitre-Glo assay ordered leftto right in the same order as in the graphical display of CellTitre-Glodata at upper right.

FIG. 85 shows measurement of metabolic stability of SW054384 byincubation with murine liver S9 microsomes. Measured half-life is 21.72minutes. SW054384 (2 mM in DMSO) was incubated with Murine S9 (Lot KWB)fraction and Phase I (NADPH Regenerating System) cofactors for 0-240minutes. Reactions were quenched with a 1 mL (1:1) of MeOH/(+)IS/0.2%Formic Acid, vortexed for 15 seconds, incubated at RT for 10 minutes andspun for 5 minutes at 2400 rpms. Supernatant (1 mL) was then transferredto an eppendorf tube and spun in a table top, chilled centrifuge for 5minutes at 13.2K rpms. Supernatant (800 μL) was transferred to an HPLCvial (w/out insert) and analyzed by HPLC/MS. A: dH₂O+0.1% FA B:MeOH+0.1% FA

FIG. 86 shows measurement of metabolic stability of SW125991 byincubation with murine liver S9 microsomes. Measured half-life is 204minutes. SW125991 (2 mM in DMSO) was incubated with Murine S9 (Lot KWB)fraction and Phase I (NADPH Regenerating System) cofactors for 0-240minutes. Reactions were quenched with 1 mL (1:1) of methanol containing0.2% formic acid and 100 ng/ml IS (IS final conc.=50 ng/ml). Sampleswere vortexed for 15 seconds, incubated at RT for 10 minutes and spunfor 5 minutes at 2400 rpm. Supernatant (1 mL) was then transferred to aneppendorf tube and spun in a table top, chilled centrifuge for 5 minutesat 13.2K rpm. Supernatant (800 μL) was transferred to an HPLC vial(w/out insert). Analyzed by Qtrap 3200 mass spectrometer.

FIG. 87 shows the structures of additional analogues of SW054384.

FIGS. 88-90 show graphs that show the level of activity in inducing the15-PGDH-luciferase fusion reporter in three different test cell linebackgrounds, V9m, LS174T, and V503. Each compound was tested at twoconcentrations, 2.5 μM, and 7.5 μM. Y-axis is luciferase activity.Compounds active in inducing the 15-PGDH-luciferase reporter include(but are not limited to): SW207997, SW207998, and SW207999.

FIG. 91 shows activity of tested compounds in inhibiting enzymaticactivity of 15-PGDH when tested at high concentrations of 2.5 uM and 7.5uM. SW207997, SW207998, and SW207999 show lesser inhibitory activity athigh concentration than the lead 15-PGDH activator, SW054384.

FIG. 92 reprises structures of 15-PGDH activators SW054383, SW125991,SW207997, SW207998, SW207999.

FIG. 93 shows graphical display of the activities of SW054383, SW125991,SW207997, SW207998, SW207999 in lowering PGE₂ levels in medium of A549cells that have been treated with 2.5 μM of each compound for 24 hours,along with addition of 2.5 ng/ml IL1-beta for the last 16 hours of theincubation, and with then collection and assay of PGE₂ concentration inthe medium at the 24 hour time point. Activity of compounds in lowering15-PGDH in the IL1-beta stimulated A549 cells is consistent with thesecompounds activating intracellular 15-PGDH.

FIG. 94 shows titration curves of 15-PGDH activator compounds in anassay measuring effects on PGE₂ levels in the medium of A549 cells thathave been stimulated with IL1-beta in the same experimental designdescribed for FIG. 93 . At 100 nM concentration of drug, the greatestreduction in levels of PGE₂ in the medium are achieved by treating cellswith SW207997 or with SW207998, followed by SW125991. SW054384 andSW207999 attain comparable levels of reduction of PGE₂ in the medium atdoses of between 0.5 μM-1.0 μM.

FIG. 95 shows assessment of toxicity of SW125991 by testing effect ofincreasing doses on colony formation of A549 cells, Vaco9M (V9m) cells,LS174T cells, and Vaco503 (V503) cells. No reduction of colony formingactivity is seen at doses up to 7.5 μM compound.

FIG. 96 shows assessment of toxicity of SW207997 by testing effect ofincreasing doses on colony formation of A549 cells, Vaco9M (V9m) cells,LS174T cells, and Vaco503 (V503) cells. No reduction of colony formingactivity is seen at doses up to 7.5 μM compound.

FIG. 97 shows assessment of toxicity of SW207998 by testing effect ofincreasing doses on colony formation of A549 cells, Vaco9M (V9m) cells,LS174T cells, and Vaco503 (V503) cells. No reduction of colony formingactivity is seen at doses up to 7.5 μM compound.

FIG. 98 shows assessment of toxicity of SW207999 by testing effect ofincreasing doses on colony formation of A549 cells, Vaco9M (V9m) cells,LS174T cells, and Vaco503 (V503) cells. No reduction of colony formingactivity is seen at doses up to 7.5 μM compound.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. All patents, publications andreferences cited in the foregoing specification are herein incorporatedby reference in their entirety.

1. A method of treating gastrointestinal diseases associated withinflammation in a subject in need thereof, the method comprising:administering to the subject a therapeutically effective amount of a15-PGDH inhibitor; wherein the 15-PGDH inhibitor comprises a compoundhaving formula (III):

wherein n is 1 or 2; R₁ is a C₁₋₈ alkyl, which is linear, branched, orcyclic and which is unsubstituted or substituted; R₂ and R₃ are the sameor different and are each selected from the group consisting of a H, alower alkyl group, (CH₂)_(n1)OR′, CF₃, CH₂—CH₂X, O—CH₂—CH₂X,CH₂—CH₂—CH₂X, O—CH₂—CH₂X, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′; Z₁is S; X₂ is N or C; R₆ and R₇ are optional and if present are the sameor different and are each selected from the group consisting of a H, F,Cl, Br, I, a lower alkyl group, (CH₂)_(n1)OR′, CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂X, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′; substituted or unsubstitutedaryl, a substituted or unsubstituted cycloalkyl, and a substituted orunsubstituted heterocyclyl; or a pharmaceutically acceptable saltthereof; wherein, where present, each instance of n1 is 1, 2, or 3;wherein, where present, each instance of X is F, Cl, Br, or I; wherein,where present, each instance of R′ is H or a lower alkyl group.
 2. Themethod of claim 1, wherein the ulcer comprises at least one of a mucosalor submucosal ulcer.
 3. The method of claim 1, wherein thegastrointestinal disease comprises at least one of oral ulcers orgastrointestinal ulcers.
 4. The method of claim 1, wherein thegastrointestinal disease comprises at least one of colitis, gastritis,or cryptitis.
 5. The method of claim 1, wherein the gastrointestinaldisease comprises ulcerative colitis.
 6. The method of claim 1, whereinthe gastrointestinal disease comprises inflammatory bowel disease. 7.The method of claim 1, wherein the 15-PGDH inhibitor is administered atan amount effective to increase prostaglandin levels in blood or tissueof the subject.
 8. The method of claim 1, wherein the 15-PGDH inhibitoris administered to the subject at an amount effective to inhibit ortreat at least one of oral or gastrointestinal ulcer formation.
 9. Themethod of claim 1, wherein the 15-PGDH inhibitor is administered to thesubject at an amount effective to inhibit or treat at least one of oralor gastrointestinal inflammation.
 10. A method of treatinggastrointestinal inflammation, ulcers, or both in a subject in needthereof, the method comprising: administering to the subject atherapeutically effective amount of a 15-PGDH inhibitor; wherein the15-PGDH inhibitor comprises a compound having formula (III):

wherein n is 1 or 2; R₁ is a C₁₋₈ alkyl, which is linear, branched, orcyclic and which is unsubstituted or substituted; R₂ and R₃ are the sameor different and are each selected from the group consisting of a H, alower alkyl group, (CH₂)_(n1)OR′, CF₃, CH₂—CH₂X, O—CH₂—CH₂X,CH₂—CH₂—CH₂X, O—CH₂—CH₂X, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′; Z₁is S; X₂ is N or C; R₆ and R₇ are optional and if present are the sameor different and are each selected from the group consisting of a H, F,Cl, Br, I, a lower alkyl group, (CH₂)_(n1)OR′, CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂X, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′; substituted or unsubstitutedaryl, a substituted or unsubstituted cycloalkyl, and a substituted orunsubstituted heterocyclyl; or a pharmaceutically acceptable saltthereof; wherein, where present, each instance of n1 is 1, 2, or 3;wherein, where present, each instance of X is F, Cl, Br, or I; wherein,where present, each instance of R′ is H or a lower alkyl group.
 11. Themethod of claim 10, wherein the subject has gastrointestinal ulcers. 12.The method of claim 10, wherein the subject has at least one of colitis,gastritis, or cryptitis.
 13. The method of claim 10, wherein the subjecthas inflammatory bowel disease.
 14. The method of claim 10, wherein the15-PGDH inhibitor is administered at an amount effective to increaseprostaglandin levels in blood or tissue of the subject.
 15. The methodof claim 10, wherein the 15-PGDH inhibitor is administered to thesubject at an amount effective to inhibit or treat gastrointestinalulcer formation.
 16. The method of claim 10, wherein the 15-PGDHinhibitor is administered to the subject at an amount effective toinhibit or treat gastrointestinal inflammation.
 17. A method of treatinggastrointestinal diseases in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a 15-PGDH inhibitor.